Recombinant obligate anaerobic gram-positive bacteria

ABSTRACT

An object of the present invention is to effectively induce cancer cell apoptosis using the anti-TRAIL-R1 antibody(ies) and the anti-TRAIL-R2 antibody(ies) and to reduce the toxicity imposed on normal cells. The present invention relates to recombinant obligate anaerobic Gram-positive bacteria that include a nucleic acid encoding a fusion protein having 3 or more anti-TRAIL-R1 single-chain antibodies and/or 3 or more anti-TRAIL-R2 single-chain antibodies, in an expressible state.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/110,131, filed Jul. 7, 2016, which is a 371 of PCT Application No.PCT/JP2014/084038, filed Dec. 24, 2014, which claims the benefit ofJapanese Patent Application No. 2014-003441, filed Jan. 10, 2014, thecontents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an antitumor agent and a marker fortumor detection comprising recombinant obligate anaerobic Gram-positivebacteria as an active ingredient.

BACKGROUND INFORMATION

A TNF-related apoptosis-inducing ligand (TRAIL) belongs to the TNFsuperfamily, and is a protein that induces apoptosis in various types ofcancer cells. TRAIL forms a trimer in vivo and binds to a TRAIL receptor(e.g., TRAIL-R1 or TRAIL-R2) containing a death domain in itsintracellular region. When TRAIL binds to TRAIL receptors, theyaggregate with each other to form a trimer, and apoptotic signals arethen transmitted intracellularly. Examples of known TRAIL receptorsinclude TRAIL-R3, TRAIL-R4, and a soluble receptor (i.e.,osteoprotegerin), in addition to the TRAIL-R1 and TRAIL-R2 describedabove (FIG. 1). TRAIL-R3, TRAIL-R4, and osteoprotegerin completely orpartially lack death domains, and such receptors do not induce apoptosiseven if TRAIL binds thereto. Thus, such receptors are referred to as“decoy receptors.”

TRAIL is less likely to cause apoptosis of normal cells. Thus, thedevelopment thereof as an antitumor agent has been in progress. In orderto efficiently induce cancer cell apoptosis with the use of TRAIL,however, it is necessary to develop a system in which TRAIL efficientlybinds to TRAIL-R1 and TRAIL-R2 without binding to the decoy receptors todeliver apoptotic signals to the cells. Agonistic antibodies againstTRAIL-R1 and TRAIL-R2 can induce apoptosis more effectively than TRAIL,since they do not bind to the decoy receptors, and administrationintervals of such antibodies may be longer, since the blood half-lifethereof is longer. Accordingly, such antibodies have been subjected toclinical trials (Non-Patent Document 1). However, since HGS-ETR1(anti-hTRAIL (human TRAIL)-R1 agonistic antibody) and HGS-ETR2(anti-hTRAIL-R2 agonistic antibody) are divalent antibodies, theseantibodies cannot induce trimer formation of TRAIL receptors, withoutcross-linking of antibodies by NK cells or macrophages having Fcreceptors (FIG. 2a ). Thus, remarkable therapeutic effects could not beattained through clinical trials of these antibodies (Non-PatentDocument 2).

HGS-TR2J (KMTR2) is known as a potent agonistic antibody that allowshTRAIL-R2 molecules on cancer cell membranes to directly aggregate anddeliver apoptotic signals without the aid of NK cells or macrophages(Non-Patent Document 3) (FIG. 2b ). It is also known that a trimer,tetramer, or pentamer of the llama-derived single-chain VHH antibodyagainst hTRAIL-R2 would very strongly induce apoptosis of cancer cellsexpressing hTRAIL-R2 without the aid of NK cells or macrophages (PatentDocument 1) (FIG. 3). In contrast, hTRAIL-R1 and hTRAIL-R2 are expressedin various normal human tissues as well as cancer cells. In particular,normal human hepatic cells are sensitive to hTRAIL and the hTRAIL-Ragonistic antibody (Non-Patent Document 4 and Non-Patent Document 5).Thus, the hTRAIL-R agonistic antibody is considered to cause hepaticdisorders as side effects.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: PCT WO/2011/098520

Non-Patent Documents

-   Non-Patent Document 1: Ghobrial et al., 2005, CA. Cancer J. Clin.,    55 (3), pp. 178-194-   Non-Patent Document 2: Micheau et al., 2013, Br. J. Pharmacol., 169    (8), pp. 1723-1744-   Non-Patent Document 3: Motoki et al., 2005, Clin. Cancer Res., 11    (8), pp. 3126-3135-   Non-Patent Document 4: Jo et al., 2000, Nat. Med., 6 (5), pp.    564-567-   Non-Patent Document 5: Mori et al., 2004, Cell Death Differ., 11    (2), pp. 203-207

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

It is an object of the present invention to induce effective cancer cellapoptosis using the anti-TRAIL-R1 antibody(ies) or the anti-TRAIL-R2antibody(ies), and to reduce the toxicity imposed on normal cells.

Means for Solving Problem

The present inventors have conducted concentrated studies in order tosolve the above problem. As a result, the present inventors prepared arecombinant Bifidobacterium strain expressing and secretinganti-hTRAIL-R2 VHH antibody(ies) having potent agonistic activity, anddiscovered that intravenous administration of the Bifidobacterium straininduced cancer cell apoptosis at tumor foci. In addition, the presentinventors obtained a novel anti-hTRAIL-R1 VHH antibody capable ofrecognizing hTRAIL-R1 molecules on the cancer cell membrane. Accordingto the present invention, cancer cell apoptosis can be effectivelyinduced while reducing the toxicity imposed on normal cells via topicaladministration of the anti-hTRAIL-R1 antibody or the anti-hTRAIL-R2antibody having potent agonistic activity.

The present invention is based on the results of the studies describedabove, and provides the following embodiments.

(1) Recombinant obligate anaerobic Gram-positive bacteria comprising anucleic acid encoding a fusion protein comprising a signal peptide and 3or more anti-TRAIL-R1 single-chain antibodies and/or 3 or moreanti-TRAIL-R2 single-chain antibodies, in an expressible state.(2) The recombinant obligate anaerobic Gram-positive bacteria of (1),wherein the obligate anaerobic Gram-positive bacteria belong to thegenus Bifidobacterium.(3) The recombinant obligate anaerobic Gram-positive bacteria of (1) or(2), wherein the anti-TRAIL-R1 single-chain antibody comprises CDR1comprising the amino acid sequence as shown in SEQ ID NO: 22, CDR2comprising the amino acid sequence as shown in SEQ ID NO: 23, and CDR3comprising the amino acid sequence as shown in SEQ ID NO: 24.(4) The recombinant obligate anaerobic Gram-positive bacteria of any of(1) to (3), wherein the anti-TRAIL-R2 single-chain antibody comprisesCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 15, CDR2comprising the amino acid sequence as shown in SEQ ID NO: 16, and CDR3comprising the amino acid sequence as shown in SEQ ID NO: 17.(5) The recombinant obligate anaerobic Gram-positive bacteria of any of(1) to (4), wherein the fusion protein further comprises one or morefunctional peptides.(6) The recombinant obligate anaerobic Gram-positive bacteria of (5),wherein the functional peptides comprises a labeling protein.(7) An antitumor agent comprising, as an active ingredient, therecombinant obligate anaerobic Gram-positive bacteria of any of (1) to(6).(8) An anti-TRAIL-R1 antibody comprising CDR1 comprising the amino acidsequence as shown in SEQ ID NO: 22, CDR2 comprising the amino acidsequence as shown in SEQ ID NO: 23; and CDR3 comprising the amino acidsequence as shown in SEQ ID NO: 24.

This description includes all or part of the contents disclosed in thedescription and/or drawings of Japanese Patent Application No.2014-003441, to which the present application claims priority.

According to the present invention, cancer cell apoptosis can beeffectively induced while reducing the toxicity imposed on normal cellsvia topical administration of the anti-hTRAIL-R1 antibody(ies) and theanti-hTRAIL-R2 antibody(ies) having potent agonistic activity to tumorfoci.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows the structures of TRAIL and receptors thereofand signal transmission for apoptosis induction.

FIG. 2 schematically shows differences in terms of intracellulartransmission of apoptotic signals between a general antibody “a” againstTRAIL-R and an antibody “b” having agonistic activity. (In the case of ageneral antibody, crosslinking by NK cells or macrophages “c” isrequired for TRAIL-mediated apoptosis. In the case of the KMTR2antibody, crosslinking by NK cells or macrophages is not required forTRAIL-mediated apoptosis.)

FIG. 3 schematically shows that a trimer, tetramer, or pentamer of thesingle-chain VHH antibody (VHH4 in FIG. 3) induces apoptosis of cancercells expressing hTRAIL-R without crosslinking by NK cells ormacrophages.

FIG. 4 shows SDS-PAGE (SDS polyacrylamide gel electrophoresis) imagesfor purified hTRAIL-R1:Fc (a), hTRAIL-R2:Fc (b (1)), and mTRAIL (mouseTRAIL)-R2:Fc (b (2)).

FIG. 5 shows an SDS-PAGE image for the purified 4E6 monomer. An arrowindicates the 4E6 monomer.

FIG. 6 shows binding activity of the 4E6 monomer to the hTRAIL-R2:Fcantigen measured by ELISA.

FIG. 7 shows the results of measurement of the dissociation constantbetween the 4E6 monomer and the hTRAIL-R2:Fc antigen using BiacoreX-100.

FIG. 8 shows an SDS-PAGE image for the purified 4P6 monomer.

FIG. 9 shows the results of ELISA assays for binding specificities ofthe 4P6 monomer and 4E6 monomer.

FIG. 10 shows the results of measurement of the dissociation constantbetween the 4P6 monomer and the hTRAIL-R1:Fc antigen using BiacoreX-100.

FIG. 11 shows antagonistic activities of the 4P6 monomer and 4E6monomer.

FIG. 12 schematically shows the gene structure of the 4E6 dimer toxin(a), the 4E6 dimer EGFP (b), and the 4E6 tetramer (c) expressed in E.coli.

FIG. 13 shows SDS-PAGE images for purified recombinant proteins (a: 4E6dimer toxin; b: 4E6 dimer EGFP; and c: 4E6 tetramer) expressed in E.coli.

FIG. 14 shows activity of recombinant proteins (a: 4E6 dimer toxin and4E6 dimer EGFP; and b: 4E6 monomer and 4E6 tetramer) to induce apoptosisof human colon cancer (Colo205) cells.

FIG. 15 shows fluorescent staining of cancer cells (a: Colo205 cells;and b: BxPC-3 cells) with the 4E6 dimer EGFP. The vertical axisindicates the cell count and the horizontal axis indicates thefluorescence intensity of the 4E6 dimer EGFP.

FIG. 16 shows activity of the anti-hTRAIL-R2 VHH antibody tetramer (4E6tetramer) expressed in E. coli to induce apoptosis of human colon cancer(Colo205) cells (a) and pancreatic cancer (BxPC-3) cells (b).

FIG. 17 shows SDS-PAGE images for the 4E6 tetramer and the 4E6 dimerEGFP purified from 1 ml of the culture supernatant of Bifidobacterium.The results for the 4E6 tetramer are obtained from 3 clones and theresults for the 4E6 dimer EGFP are obtained from 2 clones.

FIG. 18 shows activity of the 4E6 tetramer purified from the culturesupernatant of Bifidobacterium to induce apoptosis of Colo205 cancercells.

FIG. 19 shows the antitumor effects of the 4E6 tetramer-secretingBifidobacterium in nude mice into which Colo205 cells were transplanted.The tumor volume (mm³) is shown as the mean+standard error (n=6).

FIG. 20 shows changes in the body weight of nude mice into which Colo205cells were transplanted upon administration of 4E6 tetramer-secretingBifidobacterium. The body weight is shown as the mean+standard deviation(n=6).

FIG. 21 shows the antitumor effects of 4E6 tetramer-secretingBifidobacterium in nude mice into which BxPC-3 cells were transplanted.The tumor volume (mm³) is shown as the mean+standard error (n=6).

FIG. 22 shows changes in the body weight of nude mice into which BxPC-3cells were transplanted upon administration of 4E6 tetramer-secretingBifidobacterium. The body weight is shown as the mean+standard deviation(n=6).

FIG. 23 shows an SDS-PAGE image for the purified recombinant protein(4P6 trimer) expressed in E. coli.

FIG. 24 shows activity of the anti-hTRAIL-R1 VHH antibody trimer (4P6trimer) expressed in E. coli to induce apoptosis of human colon cancer(Colo205) cells.

FIG. 25 shows an SDS-PAGE image for the 4P6 trimer purified from theculture supernatant of Bifidobacterium.

FIG. 26 shows activity of the anti-hTRAIL-R1 VHH antibody trimer (4P6trimer) purified from the culture supernatant of Bifidobacterium toinduce apoptosis of human colon cancer (Colo205) cells (a) andpancreatic cancer (BxPC-3) cells (b).

FIG. 27 shows antitumor effects of 4P6 trimer-secreting Bifidobacteriumin nude mice into which BxPC-3-Luc#2 cells were transplanted. The tumorvolume (mm³) is shown as the mean+standard error (n=5).

FIG. 28 shows changes in the body weight of nude mice into whichBxPC-3-Luc#2 cells were transplanted upon administration of 4P6trimer-secreting Bifidobacterium. The body weight is shown as themean+standard error (n=5).

MODES FOR CARRYING OUT THE INVENTION

The present invention will be described below in detail.

1. Recombinant Obligate Anaerobic Gram-Positive Bacterium 1-1. Overviewand Definition

A first aspect of the present invention is a recombinant obligateanaerobic gram-positive bacterium (hereinafter, often abbreviated as“recombinant bacteria (or bacterium)”). The recombinant bacterium of thepresent invention is a drug delivery carrier containing a nucleic acidencoding a fusion protein in an expressible state.

The term “obligate anaerobic Gram-positive bacteria (or bacterium)”refers to obligate anaerobes that are classified as Gram-positivebacteria. Herein, the term “obligate anaerobes” refers to bacteria thatare also referred to as “strictly/obligately anaerobic bacteria” thatcannot grow and die at high oxygen concentrations. In a mammalian body,therefore, these bacteria can grow in a low-oxygen anaerobic state, suchas within digestive organs and mainly in the intestine, but they cannotgrow in body fluids such as blood in which dissolved oxygen is present,or in general tissues. The term “Gram-positive bacteria (or bacterium)”refers to the generic name of bacteria that are stained violet or darkblue by Gram staining. Gram-positive bacteria include, but not limitedto, bacilli, cocci, and spirilli herein. Since Gram-positive bacteria donot contain endotoxins, they do not release endotoxins after theirdeath. Therefore, Gram-positive bacteria are preferable as drug deliverycarriers of the present invention in terms of safety. Examples of therecombinant bacteria of the present invention include those of the genusBifidobacterium (hereinafter “those of the genus Bifidobacterium” arereferred to collectively as “Bifidobacterium” as a generic name) andthose of the genus Clostridium. A preferable example thereof isBifidobacterium. This is because Bifidobacterium does not secreteexotoxin, it is in daily use as a lactic acid bacterium, and it has beenconfirmed to be safe, for example, for human bodies. Bifidobacterium maybe of any species. Preferable examples thereof are species that inhabitthe human intestine, in particular, B. bifidum, B. longum, B. brave, B.infantis, and B. adolescentis.

1-2. Configuration

The obligate anaerobic gram-positive bacterium of the present inventioncontains a nucleic acid encoding a fusion protein (hereinafter, oftenreferred to as “fusion gene”) in an expressible state. Hereinafter, thefusion gene, by which the recombinant bacterium of the present inventionis characterized, and the configuration of an expression cassette thatenables the expression of the fusion gene are specifically described.

1-2-1. Configuration of Fusion Gene

The term “nucleic acid encoding a fusion protein” (or “fusion gene”) asused herein refers to a foreign nucleic acid encoding a fusion proteinthat is constructed by the fusion of a plurality of genes, etc., usinggene-recombination technology. The fusion gene is inserted into anexpression cassette described later which is introduced into therecombinant bacteria of the present invention.

The term “fusion protein” as used herein refers to an extracellularsecretory protein containing signal peptide(s), 3 or more single-chainantibodies, and, optionally, one or more functional peptides, which arelinked. The signal peptide(s), the single-chain antibodies, and thefunctional peptides may be directly linked or indirectly linked vialinker peptides. The length and the amino acid sequence of a linkerpeptide are not particularly limited, as long as it does not inhibit thefunctions of the single-chain antibodies and the functional peptides. Apreferred example of the linker is an amino acid sequence that has alength of 20 amino acids or less or 15 amino acids or less and is notself-folded. Examples of linker peptides that can be used in the presentinvention include the IEGRMD linker peptide (SEQ ID NO: 27) and the(GGSGG)₂ linker peptide (SEQ ID NO: 28). Hereinafter, such a signalpeptide, single-chain antibodies, and functional peptides that compose afusion protein are specifically described.

(1) Signal Peptide

A signal peptide is required for the extracellular transfer of a proteinthat is biosynthesized within cells. In general, the signal peptidecomprises positively charged amino acids such as Lys and Arg on theN-terminal side followed by highly hydrophobic amino acids such as Ala,Leu, Val, Ile, Val, and Phe. Moreover, the signal peptide may contain,on the C-terminal side thereof, an insertion sequence (following thesignal sequence), which facilitates the cleavage of the signal peptideand secretion and/or an amino acid sequence comprising a site recognizedby a signal peptidase cleaving the signal peptide from the fusionprotein. The signal peptide plays a role in extracellularly secretingthe fusion protein that is expressed within the bacteria of the presentinvention via a translocator or the like that exists on the membrane.The amino acid sequence of the signal peptide is not particularlylimited. The amino acid sequences of any known signal sequences capableof functioning within obligate anaerobic Gram-positive bacteria can beused herein. Also, the amino acid length of a signal peptide is notparticularly limited. In general, the length may range from 3 to 60amino acids. A signal peptide with a short amino acid length ispreferable, because, in such case, the molecular weight of the fusionprotein is not too large.

The signal peptide is positioned on the N-terminal side of the fusionprotein.

(2) Single-Chain Antibody

The above fusion protein contains 3 or more single-chain antibodies. Theterm “single-chain antibody” as used herein refers to an antibody thatis composed of a single-chain polypeptide, and is able to recognize andbind to a target substance alone. An antibody composed of two or morechains is too large in terms of molecular weight, and thus such anantibody is unlikely to be expressed and an appropriate threedimensional structure having antibody functions is unlikely to beconstructed within obligate anaerobic Gram-positive bacteria. Therefore,the single-chain antibody of the present invention is preferably alow-molecular-weight antibody having a molecular weight of 35 kDa orless per molecule. The single-chain antibody may be either a naturalantibody or an artificial antibody.

The single-chain antibody according to the present invention istypically comprises a variable region consisting of a complementaritydetermining region (CDR) and a framework region (FR). A complementaritydetermining region is a variable region that imparts binding specificityto an antibody. In contrast, a framework region is a relativelyconserved region within a variable region. A complete variable domaincomprises 4 FRs linked with 3 CDRs. Three CDRs are referred to as CDR1,CDR2, and CDR3 in order from the N terminus, and 4 FRs are referred toas FR1, FR2, FR3, and FR4 in order from the N terminus. In a variableregion, accordingly, CDRs and FRs are positioned in the order of FR1,CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino acid terminus towardthe carboxy terminus.

The term “natural antibody” as used herein refers to an antibody havingthe same amino acid sequence as that of an antibody that is produced byany vertebrate. A specific example of a natural single-chain antibody isa single-chain antibody that is produced by animals of the familyCamelidae (Hamers-Casterman C., et al., 1993, Nature, 363: 446-448)(hereinafter, referred to as a “single-chain antibody of the familyCamelidae”). The single-chain antibody of the family Camelidae iscomposed of an H chain alone (without an L chain), it is able to bind toan antigen through the V_(H) region of its H chain alone, and themolecular weight thereof is thus about 14 kDa, which is only aboutone-tenth the molecular weight of a general antibody. Moreover, thesingle-chain antibody of the family Camelidae generally has high antigenaffinity, and it is characterized by high resistance to heat, acid, andbase (Deffar, K., et al., 2009, African Journal of Biotechnology 8 (12):2645-2652). Therefore, a single-chain antibody of the family Camelidaeis very preferable as a single-chain antibody in the present invention.Any animal species of the family Camelidae can be used herein, as longas it can produce a single-chain antibody of the family Camelidae. Forexample, antibodies from any animal species such as lamas, alpacas, andcamels can be used.

The term “artificial antibody” as used herein refers to an artificiallyconstructed antibody. Examples thereof include a single-chain antibodyprepared by introducing appropriate mutation(s) into the amino acidsequence of the aforementioned natural antibody as well as astructurally modified single-chain antibody that does not exist inprinciple in nature. Specific examples of such artificial antibodyinclude a chimeric antibody, a humanized antibody, a single-chain Fv(scFv: a single-chain fragment of a variable region) (Pierce Catalog andHandbook, 1994-1995, Pierce Chemical Co., Rockford, Ill.), a diabody, atriabody, and a tetrabody.

A “chimeric antibody” refers to an antibody whose variable region andconstant region are derived from different animal species. A chimericantibody can be produced in accordance with a conventional technique,for example, by linking a nucleic acid encoding an antibody V regionwith a nucleic acid encoding a human antibody C region, incorporatingthe resultant into an expression vector, and introducing the expressionvector into a host.

A “humanized antibody” is a modified antibody also referred to as areshaped human antibody. A humanized antibody is constructed by graftingCDRs of an antibody derived from an immunized animal onto acomplementarity determining region of a human antibody. A general generecombination technique therefor is also known.

A single-chain Fv is a synthetic antibody having a molecular weight ofabout 35 kDa or less and a structure in which a polypeptide chaincontains variable regions in the L and H chains of an immunoglobulinmolecule (that is, V_(L) and V_(H), respectively), which are linked viaa flexible linker with a sufficient length. Both variable regions canself-assemble to form a functional antigen binding site in asingle-chain Fv.

The above fusion protein needs to comprise 3 or more single-chainantibodies, so as to allow TRAIL-R to aggregate and form a trimer. Asthe number of antibodies increases, however, the molecular weight of thefusion protein becomes larger. In general, accordingly, the number ofsingle-chain antibodies is preferably several, such as 3 to 6, 3 to 5,or 3 or 4. Individual single-chain antibodies are desirably linked viaappropriate linker peptide(s). The length and the amino acid sequence ofa linker peptide are not particularly limited, as long as it does notinhibit the antigen binding activity of each single-chain antibody.

The above fusion protein comprises 3 or more single-chain antibodiesthat recognize the same antigen. When hTRAIL-R1 is an antigen, forexample, the fusion protein comprises 3 or more single-chain antibodiesthat recognize the same hTRAIL-R1.

In the present invention, the term “valency” of the antibody refers tothe number of antigen-binding sites of a single antibody molecule. Forexample, IgG is a divalent antibody having two antigen-binding sites ina single molecule. The single-chain antibody is a monovalent antibodyhaving one antigen-binding site in a single molecule. Since the fusionprotein of the present invention comprises 3 or more single-chainantibodies, the fusion protein is tri- or more valent as a whole.

The order of single-chain antibodies and functional peptide(s) describedlater is not particularly limited, as long as the antibodies arepositioned on the C-terminal side of a signal peptide in a fusionprotein. Single-chain antibodies are preferably positioned on theN-terminal side of functional peptide(s).

In the present invention, a target substance of a single-chain antibodyis TRAIL-R1 or TRAIL-R2. As described above, the recombinant bacteria ofthe present invention can grow only in anaerobic environments.Accordingly, cells in an anaerobic environment in vivo are preferabletarget cells. Specifically, preferable examples thereof include tumorcells and intestinal epithelial cells. Hereafter, the anti-TRAIL-R1antibody and the anti-TRAIL-R2 antibody are described in detail.

(i) Anti-TRAIL-R1 Antibody

In the present invention, the term “anti-TRAIL-R1 single-chain antibody”refers to a single-chain antibody against TRAIL-R1 (TNF-relatedapoptosis-inducing ligand receptor 1; TRAIL receptor 1). Theanti-TRAIL-R1 single-chain antibody used in the present invention is notparticularly limited, provided that it can specifically bind to TRAIL-REAn example of an anti-TRAIL-R1 single-chain antibody that can be used inthe present invention is 4P6, which was obtained and used in theexamples of the present specification. 4P6 is a single-chain VHHantibody derived from alpaca, and comprises CDR1 comprising the aminoacid sequence as shown in SEQ ID NO: 22, CDR2 comprising the amino acidsequence as shown in SEQ ID NO: 23, and CDR3 comprising the amino acidsequence as shown in SEQ ID NO: 24. The sequence of the framework regionof the anti-TRAIL-R1 single-chain antibody used in the present inventionis not particularly limited. For example, the sequence of a frameworkregion of the alpaca-derived single-chain antibody described in Maass D.R., et al., 2007, J. Immunol. Methods, 324: 13-25 (e.g., Clone A02described therein) can be used. Accordingly, the anti-TRAIL-R1single-chain antibody may comprise for example, FR1 comprising the aminoacid sequence as shown in SEQ ID NO: 18, FR2 comprising the amino acidsequence as shown in SEQ ID NO: 19, FR3 comprising the amino acidsequence as shown in SEQ ID NO: 20, and 1-R4 comprising the amino acidsequence as shown in SEQ ID NO: 21, although the constitution is notlimited thereto.

It is particularly preferable that the fusion protein used in thepresent invention have agonistic activity of binding to TRAIL-R1 andinducing apoptosis. TRAIL-R1 induces apoptosis upon aggregation to formtrimer or larger multimer. In order to activate TRAIL-R1, accordingly,it is particularly preferable that the fusion protein comprise 3 ormore, 4 or more, 5 or more, or 6 or more, for example, 3, 4, 5, or 6anti-TRAIL-R1 single-chain antibodies, as described above.

One aspect of the present invention relates to the anti-TRAIL-R1antibody comprising CDR1 comprising the amino acid sequence as shown inSEQ ID NO: 22, CDR2 comprising the amino acid sequence as shown in SEQID NO: 23, and CDR3 comprising the amino acid sequence as shown in SEQID NO: 24. The sequence of the framework region of the antibody is notparticularly limited. For example, the sequence described in theliterature of Maass D. R. may be employed. That is, the antibody maycomprise 1-R1 comprising the amino acid sequence as shown in SEQ ID NO:18, FR2 comprising the amino acid sequence as shown in SEQ ID NO: 19,FR3 comprising the amino acid sequence as shown in SEQ ID NO: 20, andFR4 comprising the amino acid sequence as shown in SEQ ID NO: 21. Atrivalent or larger antibody is preferable in order to activate TRAIL-REThe antibody may be an artificial antibody as described above, such as achimeric or humanized antibody.

Since an agonistic antibody against TRAIL-R1 induces apoptosis viaTRAIL-R1, the antibody of the present invention can be used as aninducer of apoptosis.

(pii) Anti-TRAIL-R2 Antibody

In the present invention, the term “anti-TRAIL-R2 single-chain antibody”refers to a single-chain antibody against TRAIL-R2 (TNF-relatedapoptosis-inducing ligand receptor 2; TRAIL receptor 2). Theanti-TRAIL-R2 single-chain antibody used in the present invention is notparticularly limited, provided that it can specifically bind toTRAIL-R2. An example of an anti-TRAIL-R2 single-chain antibody that canbe used in the present invention is 4E6, which is described in WO2011/098520. 4E6 is a single-chain VHH antibody derived from llama, andcomprises CDR1 comprising the amino acid sequence as shown in SEQ ID NO:15, CDR2 comprising the amino acid sequence as shown in SEQ ID NO: 16,and CDR3 comprising the amino acid sequence as shown in SEQ ID NO: 17.

It is particularly preferable that the fusion protein used in thepresent invention have agonistic activity of binding to TRAIL-R2 andinducing apoptosis. TRAIL-R2 induces apoptosis upon aggregation to formtrimer or larger multimer. In order to activate TRAIL-R2, accordingly,it is particularly preferable that the fusion protein comprise 3 ormore, 4 or more, 5 or more, or 6 or more, for example, 3, 4, 5, or 6anti-TRAIL-R2 single-chain antibodies, as described above.

Since an agonistic antibody against TRAIL-R2 induces apoptosis viaTRAIL-R2, the antibody of the present invention can be used as aninducer of apoptosis.

(3) Functional Peptide

The above fusion protein optionally contains one or more functionalpeptides. The term “functional peptide” used herein refers to a peptidehaving, in a living body or within cells, specific bioactivity such asenzymatic activity, catalytic activity, functions as a substrate, orbiological inhibitory or enhancement activity (for example, cytotoxicactivity). Specific examples thereof include a fluorescent protein or aluminescent protein, an enzyme, and an exotoxin.

Functional peptides may be derived from any biological species.Moreover, functional peptides may be either natural or unnatural. Theterm “natural functional polypeptide” refers to a peptide that exists innature. On the other hand, the term “unnatural functional polypeptide”refers to a modified peptide prepared by introducing appropriatemutation(s) (such as the addition, deletion, and/or substitution of anamino acid(s)) into the amino acid sequence based on the amino acidsequence of a natural functional polypeptide, as long as the functionalpeptide does not lose its own unique functions.

The above fusion protein may contain 2 or more functional peptides. Whenthe above fusion protein contains a plurality of functional peptides,however, the total molecular weight of the functional peptides ispreferably 80 kDa or less, and preferably 40 kDa or less so that theoverall molecular weight of the fusion protein does not become toolarge. When the above fusion protein contains 2 or more functionalpeptides, functional peptides may be of the same type or differenttypes. Examples thereof include a functional peptide comprising anexotoxin in combination with an enzyme and an exotoxin in combinationwith a fluorescent protein or luminescent protein. When the above fusionprotein contains 2 or more functional peptides, individual functionalpeptides may be directly linked, and they are preferably linked viaappropriate linker peptide(s), so that each functional peptide is ableto efficiently exhibit its unique functions. The length and the aminoacid sequence of a linker peptide are not particularly limited, as longas it does not inhibit the functions of the functional peptide. A fusionprotein can contain two or more different functional peptides, so thatthe fusion protein can impart different functions to a target substancerecognized by a single-chain antibody.

The functional peptide(s) are preferably positioned on the C-terminalside of the above single-chain antibody, although the location is notparticularly limited, as long as it is located on the C-terminal side ofa signal peptide in a fusion protein.

Hereafter, the fluorescent protein or luminescent protein, the enzyme,and the exotoxin described above, which can function as functionalpeptides in the recombinant bacteria of the present invention, arespecifically described.

(i) Fluorescent Protein or Luminescent Protein (Labeling Protein)

The type of fluorescent protein used as a functional peptide is notparticularly limited, as long as the nucleotide sequence thereof isknown. Such a fluorescent protein may be either natural or unnatural.For the reason described above, a fluorescent protein having a shortamino acid sequence is preferable. Moreover, the excitation wavelengthand the fluorescence wavelength are not particularly limited. Thesewavelengths may be adequately selected in accordance with situation andnecessity. Specific examples of such fluorescent protein include CFP,RFP, DsRed, YFP, PE, PerCP, APC, and GFP.

Also, the type of a luminescent protein is not particularly limited, aslong as the nucleotide sequence thereof is known. As in the case of thefluorescent protein described above, a luminescent protein may be eithernatural or unnatural, and a luminescent protein having a short aminoacid sequence is preferable. A specific example of such luminescentprotein is aequorin.

(ii) Enzyme

The type of enzyme used as a functional peptide is not particularlylimited, as long as the nucleotide sequence thereof is known. Such anenzyme may be an enzyme that directly acts on a target substance or anenzyme that does not directly act thereon, but rather acts in an areaaround a target substance. Specific examples of the latter enzymeinclude luciferase and peroxidase (e.g., horseradish peroxidase) whichcontribute to luminescence. A fusion protein in which such an enzyme islinked to the above single-chain antibody can function as animmunoenzyme.

(iii) Exotoxin

The term “exotoxin” refers to a toxic protein that is secreted frombacteria. An exotoxin to be used herein may be of any type, as long asit has cytotoxic activity and the nucleotide sequence thereof is known.Examples of such an exotoxin include Pseudomonas aeruginosa toxin(Pseudomonas toxin; PT) and derivatives thereof such as exotoxin Aprepared by removing a cell adhesion domain from PT, Diphtheria toxin(DT) and derivatives thereof, and Ricin and derivatives thereof(Brinkmann U. & Pastan I., 1994, Biochimica et Biophysica Acta, 1198(1): 27-45). Pseudomonas aeruginosa exotoxin A is known to inhibitprotein synthesis by inactivating EF-2 by ADP ribosylation and toexhibit a strong cytotoxic effect, after incorporation thereof intocancer cells. A fusion protein in which an exotoxin is linked to theabove single-chain antibody can function as an immunotoxin.

1-2-2. Configuration of Expression Cassette

The term “expression cassette” as used herein refers to an expressionsystem that contains the above fusion gene and brings the fusion gene toan expressible state as a fusion protein. The term “expressible state”as used herein refers to a situation in which a fusion gene ispositioned under the control of elements required for gene expression sothat the fusion gene contained in the expression cassette is able to beexpressed within recombinant bacteria. Examples of elements required forgene expression include a promoter and a terminator.

Promoters to be used herein are not particularly limited, as long assuch promoters can be functional within recombinant bacteria. Promotersderived from obligate anaerobic Gram-positive bacteria to be used arepreferable. When B. longum (Bifidobacterium) is used as an obligateanaerobic gram-positive bacterium, an example thereof is the hup genepromoter (SEQ ID NO: 25) of B. longum. Furthermore, promoters differingin properties of expression control including an overexpressionpromoter, a constitutive promoter, a site-specific promoter, astage-specific promoter, an inducible promoter, and the like are known.A promoter to be used for an expression cassette in the presentinvention may be any promoter without particular limitation. A promotermay be adequately selected, according to need. A preferable examplethereof is an overexpression promoter or a constitutive promoter. Apromoter is positioned on the 5′ upstream of the initiation codon of theabove fusion gene in the above expression cassette.

A terminator to be used herein is not particularly limited, as long asit can terminate the transcription of a gene transcribed by the abovepromoter within recombinant bacteria. An example of such a terminatorincludes the histone-like protein terminator (HUT) (SEQ ID NO: 26). Aterminator to be used herein is preferably a terminator derived from thesame biological species as that of a promoter, and it is more preferablya terminator that forms a pair with a promoter on the genome of thebiological species from which the promoter is derived. A terminator ispositioned on the 3′ downstream of the termination codon of the abovefusion gene, in the above expression cassette.

A vector containing an expression cassette can be introduced as anexpression vector into the bacteria to stably express the above fusionprotein within the recombinant bacteria. Alternatively, a vector may beinserted into the genome of the bacteria via homologous recombination.When an expression vector is used, a plasmid or the like can be used asa vector. A vector to be used herein is replicable within therecombinant bacteria of the present invention and contains anappropriate selection marker gene that is stably retained within thebacteria. A vector may be a shuttle vector that is replicable withinother bacteria such as Escherichia coli. Examples thereof includepKKT427, pBESAF2, and pPSAB1. When a vector is inserted into the genomeof recombinant bacteria, a fusion gene alone may be inserted into thegenome of recombinant bacteria in an expressible state. Specifically, afusion gene may be inserted under the control of an endogenous promoterand/or terminator of the recombinant bacteria.

An expression cassette may be monocistronic, which contains one fusiongene within a single expression cassette, or polycistronic, whichcontains 2 or more fusion genes.

1-3. Method for Producing Recombinant Obligate Anaerobic Gram-PositiveBacteria

The recombinant bacteria of the present invention can be produced usingmolecular genetic methods known in the art. In the case of the abovefusion gene, for example, nucleic acids each encoding a signal peptideand single-chain antibody(ies) may be constructed using techniquesdescribed in Green and Sambrook, Molecular Cloning, 4th Ed., 2012, ColdSpring Harbor Laboratory Press or Ausubel et al., Short Protocols inMolecular Biology, 3rd Ed., A compendium of Methods from CurrentProtocols in Molecular Biology, 1995, John Wiley & Sons.

The single-chain antibody can be obtained with the use of the nucleotidesequence information of the gene encoding an antibody that binds toTRAIL-R1 or TRAIL-R2. The nucleotide sequence information of theantibody may be based on, for example, the above-described nucleotidesequences of CDRs and FRs.

When a new antibody against TRAIL-R1 or TRAIL-R2 is produced, amonoclonal antibody against TRAIL-R1 or TRAIL-R2 can be prepared inaccordance with a method known in the art. A preparation example thereofis as described below.

Extracellular domains of target TRAIL-R1 or TRAIL-R2 are administered asimmunogens to animals of the family Camelidae for immunization. Ifnecessary, an adjuvant may be added for effective immunization. Examplesof an adjuvant include commercially available complete Freund's adjuvant(FCA) and incomplete Freund's adjuvant (FIA), and these adjuvants can beused alone or in combination. A single dose of an immunogen solution maycontain about 50 μg to 200 μg of the immunogen per animal above. Theintervals for immunization are not particularly limited. After primaryimmunization, an immunized animal is boosted 2 to 10 times andpreferably 5 to 7 times at intervals of several days to several weeks,and preferably 1 to 4 weeks. After primary immunization, the antibodytiter in the serum of the immunized animal is measured repeatedly byELISA (i.e., enzyme-linked immunosorbent assay) or other means.Subsequently, antibody-producing cells are collected from the immunizedanimal. Examples of antibody-producing cells include spleen cells, lymphnode cells, and peripheral blood cells, with peripheral blood cellsbeing preferable. RNA is extracted from peripheral blood cells, and cDNAis then synthesized using an oligo dT primer and a random 6-mer primer.From the cDNA, the gene of the variable region (V_(HH) region) of thesingle-chain antibody of the family Camelidae is amplified by PCR, theabove gene is incorporated into a phagemid vector such as pCANTAB6(McCafferty J. et al., 1994, Appl. Biochem. Biotech., 47, 157-173), andthe vector is then introduced into Escherichia coli TG1 byelectroporation. The above Escherichia coli TG1 is infected with anM13K07 helper phage, the resultant phages are collected, and a libraryof expression phages with a single-chain antibody variable region(V_(HH) region) of the family Camelidae is thus obtained. This isnormally a library of 1×10⁷ pfu or more.

Biopanning is performed to select phages expressing an antibody againsta target antigen. Biopanning is a method for concentrating a phagespecific to a target antigen, which involves reacting an antibody phagelibrary with an immobilized target antigen, removing unbound phages bywashing, eluting phages binding to the target antigen, infectingEscherichia coli with the phages for proliferation, and repeating theseprocedures 3 to 5 times. After re-infection of Escherichia coli TG1 withphages subjected to biopanning, TG1 clones containing V_(HH) insertedpCANTAB phagemid vectors are isolated. TG1 clones are each infected witha KO7 helper phage, and cloned phages presenting the V_(HH) antibody arethus obtained. Clones that react with the antigen are selected fromamong such phages. The nucleotide sequence of the antibody can beobtained from the phages thus obtained.

A fusion gene is inserted into the above expression cassette using amolecular genetic method so that the fusion gene can be expressed. Anexpression cassette is incorporated into a vector such as a plasmid,according to need. An expression cassette may be incorporated into avector by, for example, a method that involves cleaving the 5′ end andthe 3′ end of an expression cassette with appropriate restrictionenzyme(s) and inserting the expression cassette into a correspondingrestriction site such as a multicloning site within the vector. Further,when a vector is an expression vector that enables expression within therecombinant bacteria of the present invention, the above fusion gene canbe inserted into the expression control region (e.g., a multicloningsite between a promoter and a terminator within the vector) of theexpression vector, so that the expression cassette and the targetexpression vector can be constructed at the same time. Regardingspecific methods therefor, reference may be made to, for example, themethod described in the literature of Green and Sambrook (2012)described above.

Recombinant bacteria of interest can be produced by introducing theabove expression vector, expression cassette, or fusion gene intoobligate anaerobic Gram-positive bacteria as drug delivery carriers. Asa method for introducing an expression vector or the like into targetobligate anaerobic Gram-positive bacteria, a molecular biological methodknown in the art can be employed. For example, a known method such aselectroporation or a calcium phosphate method can be employed. Regardingspecific methods therefor, reference may be made to, for example, themethod described in the literature of Green and Sambrook (2012)described above.

2. Antitumor Agent 2-1. Overview

A second aspect of the present invention is an antitumor agent.According to the present invention, the term “antitumor agent” refers toa drug having cytotoxic activity against tumor cells, causing apoptosisof the cells, and, as a result, suppressing the proliferation of thetumor cells. The antitumor agent is required to have the beneficialeffect of suppressing tumor cell proliferation, but it is not requiredto eradicate the tumor cells.

The antitumor agent of the present invention is characterized bycomprising recombinant bacteria of the first aspect as an activeingredient. Regarding the antitumor agent of the present invention, therecombinant bacterium as an active ingredient grows only within tumorsand secretes 3 or more anti-TRAIL-R1 single-chain antibodies and/or 3 ormore anti-TRAIL-R2 single-chain antibodies within tumors, so thatapoptosis can be efficiently induced in tumors, which leads to tumorregression.

2-2. Configuration

The antitumor agent of the present invention comprises the recombinantbacteria of the first aspect as an active ingredient, as describedabove. The recombinant bacteria in this case comprises the fusion geneencoding single-chain antibodies recognizing and binding to surfaceantigens of target tumor cells (i.e., TRAIL-R1) and/or a single-chainantibodies recognizing and binding to TRAIL-R2. Therefore, therecombinant bacteria as an active ingredient of the antitumor agent ofthe present invention secrete an extracellular secretory antitumor cellfusion protein.

Tumors targeted by the antitumor agent of the present invention may beany tumors, regardless of whether they are benign or malignant, providedthat such tumors express TRAIL-R1 and/or TRAIL-R2. Examples of targettumors include brain tumors, thyroid cancer, oral cancer, esophagealcancer, gastric cancer, large bowel cancer, pharyngeal cancer, lungcancer, liver cancer, renal cancer, adrenal cancer, pancreatic cancer,biliary tract cancer, cervical cancer, uterine body cancer, ovariancancer, mammary cancer, prostate cancer, bladder cancer, fibrosarcoma,mastocytoma, and melanoma.

When the recombinant bacteria of the present invention express a fusiongene, the expression product; that is, an extracellular secretoryantitumor cell fusion protein, is secreted extracellularly. The secretedfusion protein binds to tumor cells as target substances through itssingle-chain antibody moiety, and induces apoptosis of the cells bymultimerization of TRAIL-R1 and/or TRAIL-R2.

The antitumor agent comprises the recombinant bacteria of the presentinvention as an active ingredient in a viable state. The recombinantbacteria described in the first aspect of the present invention cannotgrow and will eventually die at high oxygen concentrations. In a livingbody, accordingly, the recombinant bacterium can grow and survive onlyat sites with low oxygen partial pressure. Typical examples of suchsites include central regions of tumors (solid cancer) observed in casesof advanced cancer or within the intestine. Therefore, the antitumoragent of the present invention can be an antitumor agent delivered totumors with high selectivity when administered in vivo by injection orthe like.

Furthermore, the antitumor agent of the present invention can be used incombination with other antitumor agent(s), as long as it does notinhibit or suppress the survival and the growth of the recombinantbacteria as an active ingredient and the expression and the secretion ofthe fusion protein.

In principle, the antitumor agent of the present invention can beformulated by a method known in the art on the assumption that therecombinant bacteria are maintained or preserved in a viable state. Forexample, the method described in Remington's Pharmaceutical Sciences(Merck Publishing Co., Easton, Pa.) can be employed. Specific methodsfor formulation vary depending on methods of administration. Methods ofadministration are classified roughly into oral administration andparenteral administration. In the case of the antitumor agent of thepresent invention, parenteral administration is preferable.

When the antitumor agent of the present invention is administeredparenterally, a specific example thereof is administration by injection.When the antitumor agent of the present invention is administered viainjection, the antitumor agent can be prepared as a suspension agent bymixing the recombinant bacteria with a pharmaceutically acceptablesolvent and adding a pharmaceutically acceptable carrier, according toneed.

A “pharmaceutically acceptable solvent” may be water, apharmacologically acceptable aqueous solution other than water, or anoily fluid. Examples of an aqueous solution include a saline solutionand an isotonic solution containing glucose and other auxiliary agents.Examples of auxiliary agents include D-sorbitol, D-mannose, D-mannitol,sodium chloride, low-concentration nonionic surfactants (e.g.,Polysorbate 80™, HCO-60), and polyoxyethylene sorbitan fatty acidesters. Examples of oily fluids include sesame oil and soybean oil. Thesolvent can also be used in combination with benzyl benzoate or benzylalcohol as a solubilizing agent. Moreover, the solvent may be mixed witha buffering agent such as a phosphate buffer or a sodium acetate buffer,a soothing agent such as procaine hydrochloride, a stabilizer such asbenzyl alcohol or phenol, or an antioxidant.

An injection may be formulated by adequately mixing an active ingredientwith a pharmaceutically acceptable excipient, emulsifier, suspension,surfactant, stabilizer, pH adjusting agent, or the like in a unit dosageform required for a generally accepted pharmaceutical practice.

Examples of injection include intravascular injection, intralymphaticinjection, intramuscular injection, intraperitoneal injection, andhypodermic injection. Based on a mechanism that the recombinant bacteriaas an active ingredient of the antitumor agent of the present inventiongrow within a tumor and suppress the proliferation of such tumor cells,administration into the cardiovascular system (i.e., systemicadministration such as intravascular injection or intralymphaticinjection) is preferable when the tumor site is not identified. Examplesof intravascular injection include intravenous injection andintraarterial injection. The antitumor agent of the present inventionmay be administered intravenously or intraarterially. On the other hand,when the position of a tumor site is identified, topical administrationinvolving direct administration to a tumor may be employed, in additionto the aforementioned systemic administration.

When the antitumor agent of the present invention is administeredorally, the antitumor agent may be supplemented with a pharmaceuticallyacceptable carrier, in addition to the recombinant bacteria that is anactive ingredient.

The term “pharmaceutically acceptable carrier” refers to a substancethat facilitates the formulation of a drug or the application of a drugto a living body and is added to the drug, so as to maintain thesurvival of the recombinant bacteria as an active ingredient, whilerefraining from inhibiting or suppressing the effect of the bacteria.Examples thereof include an excipient, a binder, a disintegrator, afiller, an emulsifier, a fluid modulator to be added, and a lubricant.

Examples of an “excipient” include sugars such as monosaccharides,disaccharides, cyclodextrins, and polysaccharides (specific examplesthereof include, but are not limited to, glucose, sucrose, lactose,raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch,and cellulose), metallic salts (e.g., sodium phosphate or calciumphosphate, calcium sulfate, and magnesium sulfate), citric acid,tartaric acid, glycine, low-, medium-, and high-molecular-weightpolyethylene glycols (PEG), pluronic, and any combination thereof.

Examples of a “binder” include starch pastes prepared from corn, wheat,rice, or potato, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, andpolyvinylpyrrolidone.

Examples of a “disintegrator” include the aforementioned starches,carboxymethyl starch, crosslinking polyvinylpyrrolidone, agar, alginicacid or sodium alginate, and salts thereof.

Examples of a “filler” include the aforementioned sugars and/or calciumphosphate (e.g., tricalcium phosphate or dibasic calcium phosphate).

Examples of an “emulsifier” include a sorbitan fatty acid ester, aglycerin fatty acid ester, a sucrose fatty acid ester, and a propyleneglycol fatty acid ester.

Examples of a “fluid modulator to be added” and a “lubricant” includesilicate, talc, stearate, and polyethylene glycol.

In addition, the antitumor agent may comprise a taste and flavorcorrigent, a suspension, a diluent, a surfactant, an extending agent, ahumidifying agent, a moisturizing agent (e.g., glycerin and starch), anadsorbent (e.g., starch, lactose, kaolin, bentonite, and colloidalsilicic acid), a disintegration-suppressing agent (e.g., saccharose,stearin, cocoa butter, and hydrogenated oil), a coating agent, acolorant, a preservative, an antioxidant, aroma chemicals, a flavoringagent, a sweetening agent, and a buffering agent, according to need.One, two, or more of the carriers mentioned above may be adequatelyused, according to need.

Examples of the dosage form of an oral vaccine agent include solids(e.g., tablets, pills, sublingual agents, capsules, and drops),granules, dust formulations, powders, and solutions. Furthermore, solidscan be formulated into dosage forms via coating known in the art, suchas sugar-coated tablets, encapsulated gelatin tablets, enteric-coatedtablets, film-coated tablets, double layer tablets, and multilayertablets, according to need. Specific shapes and sizes of such dosageforms are not particularly limited, as long as the shapes and the sizesare within the scope of the relevant dosage forms known in the art.

The content of the recombinant bacteria in the antitumor agent of thepresent invention may be any amount, provided that the bacteria is ableto reach a target tumor in a viable and proliferative state via a singleadministration in principle and the bacteria would impose no orsubstantially no harmful side effects on the subject to which the agentis administered. Such contents vary depending on the types of targetcells of the antitumor agent, cancer stages, tumor sizes, the number oftumor sites throughout the body, dosage forms of the antitumor agent,and methods for administration of the antitumor agent. The content isadequately determined in view of these conditions.

Objects to which the antitumor agent of the present invention isadministered are subjects having tumors or subjects with the highpossibility of having tumors. The term “subjects” as used herein refersto animals to which the antitumor agent of the present invention isadministered. Examples thereof include mammals, preferably humans, dogs,cats, horses, mice, rats, rabbits, cattle, and monkeys, and morepreferably humans.

2-3. Effects

Regarding the antitumor agent of the present invention, the recombinantbacteria as an active ingredient survive and proliferate only withintumors with low oxygen partial pressure and secrete antitumor cellfusion proteins. Therefore, the fusion proteins can be efficientlydelivered to tumor cells and allowed to continuously act. Further, whenthe oxygen partial pressure in the tissue is increased by suppression oftumor cell proliferation and tumor regression due to the effects of thefusion proteins, the recombinant bacteria that are obligate anaerobicGram-positive bacteria become inviable, and thus such bacteria can beautomatically eliminated from the living body. Accordingly, a safer andmore convenient therapeutic method in comparison with conventionalbacteriotherapy can be provided, and it can exert antitumor effectsagainst the solid cancer via intravenous administration ofnon-pathogenic obligate anaerobic bacteria alone without any otherantitumor agents.

Since the antitumor agent of the present invention can be administeredvia intravenous injection, it is advantageous in terms of low levels ofsubject invasiveness.

3. Marker for Tumor Detection 3-1. Overview

A third aspect of the present invention is a marker for tumor detection.The term “marker for tumor detection” used in the present inventionrefers to a marker capable of detecting a tumor in vivo.

The marker for tumor detection of the present invention comprises therecombinant bacteria of the first aspect as an active ingredient. Withthe use of the marker for tumor detection of the present invention, thepositions or the sizes of tumors in vivo can be observed because therecombinant bacteria as an active ingredient grow only within tumors andsecrete an enzyme, a fluorescent protein, or a luminescent proteinwithin tumors.

3-2. Configuration

The basic configuration is in accordance with that of the antitumoragent of the second aspect. Accordingly, differences with the aboveantitumor agent are mainly explained herein, and explanations ofoverlapping configurations are omitted in principle. The marker fortumor detection of the present invention comprises the recombinantbacteria of the first aspect as an active ingredient, as describedabove. The recombinant bacterium in this case is characterized in that afusion gene encodes single-chain antibodies recognizing and binding to asurface antigen of target tumor cells, and a functional peptide encodesa labeling protein or a protein inducing labeling. Examples of alabeling protein include the fluorescent protein and the luminescentprotein described above. Examples of a protein inducing labeling includeenzymes (luciferase and peroxidase), the substrates of which areluminophores or fluorophores, such as luciferin and luminol. Therefore,the recombinant bacterium as an active ingredient of the marker fortumor detection of the present invention secretes an extracellularsecretory antitumor cell immunomarker (immunolabel).

When the recombinant bacteria of the present invention express a fusiongene, the expression product; that is, an extracellular secretoryantitumor cell immunomarker, is secreted extracellularly. The secretedimmunomarker binds to TRAIL-R1 or TRAIL-R2 on tumor cells that aretarget substances with its single-chain antibody moiety, and thus thecells are labeled. Specifically, when a functional peptide is a labelingprotein such as a luminescent protein or a fluorescent protein, tumorcells are directly labeled with the labeling protein linked with thesingle-chain antibody moiety. On the other hand, when a functionalpeptide is an enzyme, tumor cells are enzymatically labeled with theenzyme linked to the single-chain antibody moiety.

The marker for tumor detection of the present invention can be used incombination with the antitumor agent of the second aspect. In this case,the recombinant bacteria as active ingredients may be the same as ordifferent from the recombinant bacteria that secrete the marker fortumor detection and the antitumor agent separately as individualmolecules. Specifically, the recombinant bacteria may secrete differentfusion proteins containing single-chain antibodies that recognize thesame target substance. Alternatively, the recombinant bacteria maysecrete one fusion protein containing an exotoxin and a labeling proteinor an enzyme in its functional protein moiety. In such cases, the sametumor cells can be labeled and damaged by the effects of the exotoxin.

Moreover, the marker for tumor detection can be used in combination withother antitumor agents, as long as they do not inhibit or suppress thesurvival and the growth of the recombinant bacterium as an activeingredient and the expression and the secretion of the immunomarker.

3-3. Detection

When the marker for tumor detection of the present invention isadministered to a subject who has a tumor, the recombinant bacteria asan active ingredient proliferate within the tumor, and an antitumor cellimmunomarker is secreted. Tumor cells are labeled with the secretedimmunomarker. The labeled tumor cells are detected by luminescence orfluorescence emitted by the labeling protein itself, when theimmunomarker is a labeling protein such as a luminescent protein or afluorescent protein. In the case of enzymatic labeling, luminescence orfluorescence resulting from an enzymatic reaction of a substrate such asluciferin administered in vivo may be detected. Methods for detecting animmunomarker are not particularly limited. Since many tumors are presentwithin a living body, such tumors may be exposed by surgery such aslaparotomy to detect an immunomarker. An immunomarker may also bedetected noninvasively through detection of luminescence or fluorescencein vivo from outside the body. A noninvasive method involving detectionfrom outside the body is preferable. An example of such a method thatcan be used herein for detecting luminescence or fluorescence derivedfrom an immunomarker from outside the body is, but is not limited to, anin vivo bioimaging method. For example, an immunomarker can be detectedby the methods described in Katz, M. H. et al., 2003, Cancer Res. 63:5521-5525, Schmitt, C. A. et al., 2002, Cancer Cell, 1: 289-298, andKatz, M. H. et al., 2003, J. Surg. Res., 113: 151-160. Such detectioncan also be performed using a commercially available IVIS Imaging System(Caliper) or an apparatus similar thereto.

3-4. Effects

With the use of the marker for tumor detection of the present invention,the positions or the sizes of tumors in vivo can be observed fromoutside the living body based on an immunomarker. Further, with the useof the marker for tumor detection in combination with the antitumoragent of the second aspect of the present invention, the immunotoxinsuppresses tumor cell proliferation, and tumor regression or thetherapeutic effects can be monitored over time by detecting tumors invivo from outside the living body using the immunomarker.

EXAMPLES Example 1: Preparation of Anti-Human TRAIL-R2 Gene ExpressionCassette to be Expressed in Bifidobacterium (1) Gene Expression Cassetteof Anti-hTRAIL-R2 VHH Antibody Tetramer (4E6 Tetramer) to be Expressedin Bifidobacterium

A gene (SEQ ID NO: 1) was prepared via DNA synthesis using the promoterregion derived from the B. longum hup gene (SEQ ID NO: 25), the uspsecretory signal sequence derived from the B. longum (SEQ ID NO: 29),DTY (the insertion sequence following the signal sequence), the aminoacid sequence of 4E6 disclosed in WO 2011/098520, the linker peptide(GGSGG)₂ derived from the 8C7 EGFP gene (SEQ ID NO: 28), and thehistone-like protein-derived terminator (HUT) (SEQ ID NO: 26), and DNAencoding the His-Tag sequence was added to the C terminus.

(2) Gene Expression Cassette of Fusion Protein of Anti-hTRAIL-R2 VHHAntibody Dimer and Pseudomonas aeruginosa Exotoxin A Subunit (4E6 DimerToxin) to be Expressed in Bifidobacterium

A gene (SEQ ID NO: 2) was prepared via DNA synthesis using the promoterregion derived from the hup gene of B. longum (SEQ ID NO: 25), the uspsecretory signal sequence derived from the B. longum, DTY (the insertionsequence following the signal sequence) (SEQ ID NO: 29), the amino acidsequence of 4E6 disclosed in WO 2011/098520, the linker peptide (GGSGG)₂derived from the 8C7 EGFP gene (SEQ ID NO: 28), Pseudomonas aeruginosaexotoxin A (the DNA sequence of exotoxin A encoded by pJH8 (purchasedfrom ATCC)), and the histone-like protein-derived terminator (HUT) (SEQID NO: 26), and DNA encoding the His-Tag sequence added to the Cterminus.

(3) Gene Expression Cassette of Fusion Protein of Anti-hTRAIL-R2 VHHAntibody Dimer and Green Fluorescent Protein (4E6 Dimer EGFP) to beExpressed in Bifidobacterium

A DNA sequence (SEQ ID NO: 3) was prepared using the promoter regionderived from the hup gene of B. longum (SEQ ID NO: 25), the uspsecretory signal sequence derived from the B. longum (SEQ ID NO: 29),DTY (the insertion sequence following the signal sequence), and thehistone-like protein-derived terminator (HUT) (SEQ ID NO: 26), andadding a DNA sequence encoding the linker peptide (GGSGG)₂ derived fromthe 8C7 EGFP gene (SEQ ID NO: 28), the EGFP gene (Zhang G. et al., 1996,Biochem Biophys Res Commun, 227 (3):707-711), and the His-Tag sequenceto the 3′ terminus of the 4E6 dimer gene.

Example 2: Preparation of Cultured Drosophila Cells that Express andSecrete hTRAIL-R1:Fc, hTRAIL-R2:Fc, and mTRAIL-R2:Fc and Purification ofthe Recombinant Proteins (1) Preparation of Gene Expression Cassette

(1-1) Gene Expression Cassette of Human TRAIL-R1:Alpaca Fc(hTRAIL-R1:Fc) to be Expressed in Cultured Drosophila Cells

DNA comprising the KpnI site, the consensus sequence for translationinitiation (Cavener D. R., 1987, Nucleic Acids Res. 15, 1353-1361), theBip secretory signal (Life Technologies), the hTRAIL-R1 extracellularregion (Accession No. AAC51226, amino acids 109 to 239), the IEGRMDlinker (SEQ ID NO: 27), the Lama pacos (alpaca) IgG1 Fc (Accession No.AM773729, amino acids 102 to 335), the His-Tag sequence, the terminationcodon, and the XhoI site was synthesized (SEQ ID NO: 4).

(1-2) Gene Expression Cassette of Human TRAIL-R2:Alpaca Fc(hTRAIL-R2:Fc) to be Expressed in Cultured Drosophila Cells

DNA comprising the KpnI site, the consensus sequence for translationinitiation (Cavener D. R., 1987, Nucleic Acids Res., 15, 1353-1361), theBip secretory signal (Life Technologies), the hTRAIL-R2 extracellularregion (Accession No. Q6FH58, amino acids 54 to 182), the IEGRMD linker(SEQ ID NO: 27), Lama pacos (alpaca) IgG1 Fc (Accession No. AM773729,amino acids 102 to 335), the His-Tag sequence, the termination codon,and the XhoI site was synthesized (SEQ ID NO: 5).

(1-3) Gene Expression Cassette of Mouse TRAIL-R2: Alpaca Fc(mTRAIL-R2:Fc) to be Expressed in Cultured Drosophila Cells

DNA comprising the KpnI site, the consensus sequence for translationinitiation (Cavener D. R., 1987, Nucleic Acids Res. 15, 1353-1361), theBip secretory signal (Life Technologies), the mTRAIL-R2 extracellularregion (Accession No. Q9QZM4, amino acids 52 to 177), the IEGRMD linker(SEQ ID NO: 27), Lama pacos (alpaca) IgG1 Fc (Accession No. AM773729,amino acids 102 to 335), the His-Tag sequence, the termination codon,and the XhoI site was synthesized (SEQ ID NO: 6).

(2) Preparation of Cultured Drosophila Cells that Express and SecretehTRAIL-R1:Fc, hTRAIL-R2:Fc, and mTRAIL-R2:Fc and Purification of theRecombinant Proteins

In order to obtain fusion proteins of extracellular regions ofhTRAIL-R1, hTRAIL-R2, or mTRAIL-R2 and an Fc of alpaca IgG1, vectorsthat express and secrete these recombinant proteins in S2 cells, whichare cultured Drosophila cells, were constructed (i.e.,pAc5.1/hTRAIL-R1-Fc, pAc5.1/hTRAIL-R2-Fc, and pAc5.1/mTRAIL-R2-Fc). Thegene expression cassettes that express and secrete recombinant proteinsin S2 cells were inserted between the KpnI site and the XhoI site of thepAc5.1/V5-HisA plasmid (Life Technologies). The pAc5.1/V5-HisA plasmidand the pCoHygro plasmid (Life Technologies) containing a hygromycinresistance gene were introduced into the S2 cell at the ratio of 19:1 bythe calcium phosphate method. The cells were cultured in Schneider'sDrosophila Medium (Life Technologies) containing 300 μg/ml hygromycin(Life Technologies) and 10% fetal bovine serum (Tissue CultureBiologicals) to obtain the drug-resistant cells. The drug-resistantcells (1×10⁷ cells/ml) were cultured in Express Five SFM (LifeTechnologies) containing 20 mM glutamine, and the culture supernatantwas recovered after cultivating for 7 days. The recombinant proteinswere purified using TALON resin (TAKARA BIO INC.). Specifically, theculture supernatant was added to a column filled with TALON resin, thecolumn was washed with a wash buffer (25 mM HEPES, pH 7.4, 0.3 M NaCl, 5mM imidazole), and recombinant proteins were eluted with an elutionbuffer (25 mM HEPES, pH 7.4, 0.3 M NaCl, 150 mM imidazole). The purifiedproteins were subjected to SDS-polyacrylamide electrophoresis andstained with Coomassie Brilliant Blue (CBB) R-250 (Bio-Rad), so as toconfirm that the proteins had been purified (FIG. 4).

Example 3: Expression, Purification, and Binding Activity of Recombinant4E6 Monomeric Protein in E. coli BL21 (DE3)

(1) Preparation of Gene Expression Cassette of Anti-hTRAIL-R2 VHHAntibody/Myc-Tag (4E6 Monomer) to be Expressed in E. coli

On the basis of the amino acid sequence information regarding the 4E6VHH monomer disclosed in WO 2011/098520, a gene to be expressed in E.coli was prepared via DNA synthesis (SEQ ID NO: 7).

(2) Expression of 4E6 Monomer in E. coli and Purification Thereof

A vector expressing a 4E6 monomer was obtained by inserting the genecassette between the NdeI site and the NotI site of pET22b(+). Thisexpression vector (Plasmid DNA) was introduced into E. coli BL21Star™(DE3) One Shot (Life Technologies). In accordance with the attachedinstructions, recombinant E. coli cells were cultured in 100 ml of 2YTmedium containing 100 μg/ml ampicillin (Sigma-Aldrich) at 37° C., and0.5 mM IPTG (isopropyl-β-thiogalactopyranoside, TAKARA BIO INC.) wasadded thereto when OD₆₀₀ reached 0.4 to 0.5, followed by cultivation at30° C. for 3 hours. Following the cultivation, E. coli cells wererecovered and resuspended in 10 ml of an extraction buffer (50 mM Naphosphate, pH 7.8, 300 mM NaCl, EDTA-free protease inhibitor cocktail,Roche). The cells were disrupted by ultrasonication on ice usingSonifier 250 (Branson) at the output control of 2 and the duty cycle of80% for 1 minute twice. After the treatment, the suspension wascentrifuged at 15,000 rpm and 4° C. for 20 minutes, and the supernatantwas recovered. The fusion protein was purified with the use of theHisTrap column (GE Healthcare, U.K.). The centrifuged supernatant of theultrasonicated suspension was directly applied to the HisTrap column,the column was washed with a binding buffer (20 mM sodium phosphate, 0.5M NaCl, 20 mM imidazole, pH 7.4), and the fusion protein was then elutedwith an eluate containing 500 mM imidazole. The purified sample wassubjected to 15% SDS-polyacrylamide gel electrophoresis and then stainedwith CBB. The purified 4E6 monomeric protein was observed atapproximately 15 KDa, and the concentration of the 4E6 monomer wasestimated by comparing the results of staining of BSA (bovine serumalbumin). As a result, the concentration of the 4E6 monomer was found tobe about 3 μg/μl (FIG. 5).

(3) Verification of Binding Activity of 4E6 Monomer to hTRAIL-R2:FcAntigen Via ELISA

hTRAIL-R2:Fc was prepared as described in Example 2. 0.1 M NaHCO₃(Blank), 0.1 M NaHCO₃ containing 1 μg/ml or 10 μg/ml BSA (BSA negativeantigen), or 0.1 M NaHCO₃ containing 1 μg/ml or 10 μg/ml hTRAIL-R2:Fcwas added to a 96-well Immuno plate (Nunc) at 50 μl/well, and the platewas left at 4° C. overnight. SuperBlock-PBS (Thermo Scientific) wasadded thereto at 350 μl/well, and the plate was left at room temperaturefor 1 hour. The plate was washed with PBS-T (phosphate-buffered salinecontaining 0.05% Tween 20) at 400 μl/well, SuperBlock-PBS containing 1μg/ml of 4E6 monomer was added thereto at 40 μl/well, and the reactionwas allowed to proceed at room temperature for 1 hour. The plate waswashed again three times with PBS-T at 400 μl/well, the anti-Myc mousemonoclonal antibody 9E10 (Santa Cruz Biotechnology) diluted 500-foldwith SuperBlock-PBS was added thereto at 40 μl/well, and the reactionwas allowed to proceed at room temperature for 1 hour. The plate waswashed three times with PBS-T at 400 μl/well, the anti-mouse IgG goatantibody HRP was added thereto at 40 μl/well, and the plate was left atroom temperature for 1 hour. Then, the plate was washed three times withPBS-T at 400 μl/well, a TMB reagent (Wako Pure Chemical Industries,Ltd.) was added thereto at 50 μl/well, the reaction was allowed toproceed for approximately 10 minutes, and the reaction was thenterminated with 0.5 M sulfuric acid. Then, the absorbance at 450 nm wasmeasured, and the mean and the standard deviation of the measurementsperformed in triplicate were calculated. The purified 4E6 monomericprotein specifically bound to hTRAIL-R2:Fc (FIG. 6).

(4) Measurement of Dissociation Constant (K_(D)) Between 4E6 Monomer andhTRAIL-R2 ECD (Extracellular Domain)

Binding affinity between the 4E6 monomer and hTRAIL-R2:Fc (see Example2) was analyzed via surface plasmon resonance technology using BiacoreX-100 (GE Healthcare). Measurement was carried out via multi-cyclekinetics analysis in accordance with the Biacore X-100 instructions byadding the 4E6 monomer at concentrations of 0.919 nM, 1.838 nM, 3.675nM, 7.35 nM, 14.7 nM, 29.4 nM, 58.8 nM, and 117.6 nM.

FIG. 7 shows a sensorgram and fitting curves at each concentrations. TheK_(D) value between the 4E6 monomer and the recombinant humanTRAIL-R2:Fc antigen was 7.5×10⁻¹¹ M.

Example 4: Obtaining Novel Anti-hTRAIL-R1 VHH Antibody (4P6 Monomer) (1)Isolation of Novel Anti-hTRAIL-R1 VHH Antibody (4P6 Monomer) Gene

Since the anti-TRAIL-R1 VHH antibody has not been known, it was preparedin the following method. Specifically, the gene of VHH antibody thatbinds to hTRAIL-R1:human Fc (R&D Systems) was isolated via phage displaywith reference to the document of Maass et al. (J. Immunol. Methods,2007, 324, 13-25). Alpaca was immunized with 100 μg of hTRAIL-R1:alpacaFc six times at intervals of 1 to 2 weeks, leukocytes were recovered 8weeks later, and RNA was extracted using RNeasy (Qiagen, Venlo,Netherland). cDNA was synthesized from this RNA with an oligo dT primerand a random 6-mer primer using the PrimeScriptII 1^(st) strand cDNAsynthesis kit (TAKARA BIO INC.). The VHH antibody gene was amplified viaPCR using PrimeSTAR GXL DNA polymerase (TAKARA BIO INC.), and PCR wascarried out via a cycle of 95° C. for 1 minute and 25 cycles of 98° C.for 10 seconds, 55° C. for 15 seconds, and 68° C. for 1 minute. Theamplified product was subjected to a PCR procedure comprising a cycle of95° C. for 1 minute and 20 cycles of 98° C. for 10 seconds, 60° C. for15 seconds, and 68° C. for 1 minute in the same manner, so as to amplifythe antibody gene. PCR was carried out using primers having the primerDNA sequence 1 (SEQ ID NO: 8) and the primer DNA sequence 2 (SEQ ID NO:9). The sequence of the isolated antibody gene (4P6) was determined viacycle sequencing using the BigDye Terminator v3.1 (Life Technologies).As a result, the isolated antibody was found to comprise CDR1 comprisingthe amino acid sequence as shown in SEQ ID NO: 22, CDR2 comprising theamino acid sequence as shown in SEQ ID NO: 23, and CDR3 comprising theamino acid sequence as shown in SEQ ID NO: 24.

(2) Preparation of Gene Expression Cassette of 4P6 Monomer to beExpressed in Cultured Drosophila Cells

DNA comprising the KpnI site, the consensus sequence for translationinitiation (Cavener D. R., 1987, Nucleic Acids Res. 15, 1353-1361), theBip secretory signal (Life Technologies), the 4P6 gene, the His-Tagsequence, the Myc-Tag sequence, the termination codon, and the XhoI sitewas synthesized.

(3) Preparation of Cultured Drosophila Cells that Express and SecreteAnti-hTRAIL-R1 VHH Antibody Monomer (4P6 Monomer) and Purification ofthe Recombinant Protein

The gene expression cassette prepared in the above (2) was insertedbetween the KpnI site and the XhoI site of the pAc5.1/V5-HisA plasmid(Life Technologies), so as to prepare a vector (i.e., pAc5.1/4P6monomer) that allows expression and secretion of the 4P6 monomer incultured Drosophila cells (i.e., S2 cells). This plasmid and thepCoHygro plasmid containing a hygromycin resistance gene were introducedinto the S2 cells at the ratio of 19:1 by the calcium phosphate method.The cells were cultured in the Schneider's Drosophila Medium (LifeTechnologies) containing 300 μg/ml hygromycin (Life Technologies) and10% fetal bovine serum to obtain the drug-resistant cells. Thedrug-resistant cells were cultured in Express Five SFM (LifeTechnologies) containing 20 mM glutamine, and the culture supernatantwas obtained. The 4P6 monomer was purified using the HisTrap column (GEHealthcare). The purified protein was subjected to 12.5%SDS-polyacrylamide gel electrophoresis and then stained with the OrioleFluorescent Gel Stain (Bio-Rad) (FIG. 8).

Example 5: Measurement of Binding Specificity of Anti-hTRAIL-R1 VHHAntibody Monomer (4P6 Monomer) and Affinity Thereof with hTRAIL-R1 (1)Analysis of Binding Specificity of 4P6 Monomer and 4E6 Monomer Via ELISA

Whether or not the 4P6 monomer selectively binds to TRAIL-R1 wasinvestigated via ELISA. 50 μl each solutions of hTRAIL-R1:Fc,hTRAIL-R2:Fc, mTRAIL-R2:Fc (see Example 2), or bovine serum albumindissolved in 0.1 M NaHCO₃ at 1 μg/ml was added to the 96-well NuncImmuno plate (Thermo Scientific), and the resultant was left at 4° C.overnight. 300 μl of SuperBlock (TBS) Blocking Buffer (ThermoScientific) was added and the resultant was left at room temperature for1 hour. After the solutions were removed from the wells, the 4P6 monomeror 4E6 monomer dissolved at 10 μg/ml in a blocking buffer was added at50 μl/well, and the resultant was left at room temperature for 1 hour.After the plate was washed three times with PBS containing 0.05% Tween20, 50 μl of the 9E10 anti-Myc antibody (Santa Cruz Biotechnology)dissolved at 67 ng/ml in a blocking buffer was added thereto, and theresultant was left at room temperature for 1 hour. The resultant waswashed three times, 50 μl of anti-mouse IgG HRP dissolved in a blockingbuffer was added thereto, and the resultant was left at room temperaturefor 1 hour. The resultant was washed three times, 50 μl of a TMBsolution (Wako Pure Chemical Industries, Ltd.) was added thereto, andthe resultant was left at room temperature for 10 minutes. 50 μl of 0.5M sulfuric acid was added and the absorbance at 450 nm was measured. 2wells per samples were analyzed, and the average and the error of themeasured value were calculated. It was confirmed that the 4P6 monomerspecifically binds to hTRAIL-R1, and the 4E6 monomer specifically bindsto hTRAIL-R2 (FIG. 9).(2) Measurement of Dissociation Constant Between Anti-hTRAIL-R1 VHHAntibody Monomer (4P6 Monomer) and hTRAIL-R1 ECD (Extracellular Domain)

Binding affinity between the 4P6 monomer and recombinant human TRAIL-R1ECD was analyzed via surface plasmon resonance technology using BiacoreX-100 (GE Healthcare). hTRAIL-R1:Fc (see Example 2) was fixed on asensor chip (CMS) at approximately 1,000 RU. Measurement was carried outvia single-cycle kinetics analysis in accordance with the Biacore X-100instructions by successively adding the 4P6 monomer at concentrations of0.1 nM, 0.5 nM, 2.5 nM, 12.5 nM, and 62.5 nM. FIG. 10 shows a sensorgramand a fitting curve. The K_(D) value (dissociation constant) was3.4×10⁻¹¹ M.

(3) Antagonistic Activity of 4P6 Monomer and 4E6 Monomer

Whether or not the 4P6 monomer exerts antagonistic activity againsthTRAIL that binds to hTRAIL-R1 of cancer cells and induces apoptosis wasanalyzed. Human colon cancer cells (Colo205 cells) were suspended inRPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum(Tissue Culture Biologicals), and the cells were added to a Falcon96-well culture plate (Becton, Dickinson and Company) at 3×10³ cells/50μl medium/well. The cells were cultured overnight, and 50 μl each ofmedia containing TRAIL at the final concentration of 100 ng/ml, as wellas the 4P6 monomer, and the anti-hTRAIL-R2 VHH antibody (4E6 monomer) atvarious concentrations was added thereto. The cells were furthercultured overnight, 10 μl of a viable cell count reagent SF (NacalaiTesque, Inc.) was added thereto, and the absorbance at 450 nm wasmeasured after 2 hours of cultivation. The measured value of a cell-freemedium in a well was subtracted as a background value. The valuesmeasured for the wells containing cells alone were designated 100%, andthe relative values were determined. The results were shown as the meanplus the standard deviation of the results for 3 wells at eachconcentration. As shown in FIG. 11, apoptosis caused by TRAIL could notbe inhibited by the 4P6 monomer or the 4E6 monomer alone. When the 4P6monomer and the 4E6 monomer were added simultaneously, however,apoptosis was inhibited in a dose-dependent manner Since hTRAIL, the 4P6monomer, and the 4E6 monomer have substantially equivalent molecularweights, the VHH antibody at 100 ng/ml is substantially equal to hTRAILat 100 ng/ml in terms of molar concentration. The results describedabove demonstrate that the 4P6 monomer and the 4E6 monomer not only bindto hTRAIL-R1 and hTRAIL-R2 on the cell surface but also act asantagonists.

As described above, a novel antibody (4P6) capable of specificallybinding to hTRAIL-R1 and serving as an antagonist was obtained.

Example 6: Preparation of 4E6 Dimer Toxin, 4E6 Dimer EGFP, 4E6 Tetramer,and Recombinant E. coli BL21 (DE3) and Expression and Purification ofthe Recombinant Proteins (1) Preparation of Gene Expression Cassette

(1-1) Preparation of Gene Expression Cassette of Fusion Protein ofAnti-hTRAIL-R2 VHH Antibody Dimer and Pseudomonas aeruginosa Exotoxin ASubunit (4E6 Dimer Toxin) to be Expressed in E. coli

The 4E6 dimer toxin gene to be expressed in E. coli was amplified viaPCR using the gene expression cassette (SEQ ID NO: 2) of 4E6 dimer toxinto be expressed in Bifidobacterium inserted into the pBluescriptII(+)plasmid as a template, and primers having the DNA sequence 5 (SEQ ID NO:12) and the DNA sequence 6 (SEQ ID NO: 13). PCR was carried out usingPrimeSTAR GXL DNA Polymerase (TAKARA BIO INC.) as DNA polymerase througha cycle of 95° C. for 1 minute and 25 cycles of 98° C. for 10 seconds,60° C. for 15 seconds, and 68° C. for 2 minutes. The amplified productwas purified using the MinElute column (Qiagen) in accordance with theattached protocols, digested with restriction enzymes NdeI and NotI, andsubjected to 1.2% agarose gel electrophoresis. The band of interest wascleaved from the gel, followed by isolation and purification with theuse of the DNA gel extraction kit (Qiagen) in accordance with theattached protocols. The resultant was inserted between the NdeI site andthe NotI site of pET-22b(+) (Novagen), so as to construct the 4E6 dimertoxin comprising Strep-tag at its N terminus (Schmidt T. G., Skerra A.,2007, Nat. Protoc., 2 (6): 1528-1535), two VHH monomers linked with eachother with the linker peptide (GGSGG)₂ (SEQ ID NO: 28), and thePseudomonas aeruginosa exotoxin subunit A (toxin) linked to the Cterminus of the 4E6 dimer through the XbaI sequence (SerArg) (FIG. 12a).

(1-2) Preparation of Gene Expression Cassette of Fusion Protein ofAnti-hTRAIL-R2 VHH Antibody Dimer and Green Fluorescent Protein (4E6Dimer EGFP) to be Expressed in E. coli

The 4E6 dimer EGFP gene to be expressed in E. coli was amplified via PCRusing the gene expression cassette (SEQ ID NO: 3) of the 4E6 dimer toxinto be expressed in Bifidobacterium inserted into the pBluescriptII(+)plasmid as a template and primers having the DNA sequence 5 (SEQ ID NO:12) and the DNA sequence 7 (SEQ ID NO: 14). PCR was carried out usingPrimeSTAR GXL DNA Polymerase (TAKARA BIO INC.) as DNA polymerase througha cycle of 95° C. for 1 minute and 25 cycles of 98° C. for 10 seconds,60° C. for 15 seconds, and 68° C. for 2 minutes. The amplified productwas purified using the MinElute column (Qiagen) in accordance with theattached protocols, digested with restriction enzymes NdeI and NotI, andsubjected to 1.2% agarose gel electrophoresis. The band of interest wascleaved from the gel, followed by isolation and purification with theuse of the DNA gel extraction kit (Qiagen) in accordance with theattached protocols. The resultant was inserted between the NdeI site andthe NotI site of pET-22b(+) (Novagen), so as to construct the 4E6 dimerEGFP comprising Strep-tag at its N terminus, two VHH monomers linkedwith each other with the linker peptide (GGSGG)₂ (SEQ ID NO: 28), and agreen fluorescent protein (EGFP), to which the linker peptide (GGSGG)₂(SEQ ID NO: 28) was bound to the N terminus, linked to the C terminus ofthe 4E6 dimer through the XbaI sequence (SerArg) (FIG. 12b ).

(1-3) Preparation of Gene Expression Cassette of Anti-hTRAIL-R2 VHHAntibody Tetramer (4E6 Tetramer) to be Expressed in E. coli

The 4E6 tetramer gene to be expressed in E. coli was amplified via PCRusing the gene expression cassette (SEQ ID NO: 1) of the 4E6 tetramer tobe expressed in Bifidobacterium inserted into the pEX-K plasmid as atemplate and primers having the DNA sequence 3 (SEQ ID NO: 10) and theDNA sequence 4 (SEQ ID NO: 11). PCR was carried out using PrimeSTAR GXLDNA Polymerase (TAKARA BIO INC.), as DNA polymerase through a cycle of95° C. for 1 minute and 25 cycles of 98° C. for 10 seconds, 60° C. for15 seconds, and 68° C. for 2 minutes. The amplified product was purifiedusing the MinElute column (Qiagen) in accordance with the attachedprotocols, digested with restriction enzymes NdeI and NotI, andsubjected to 1.2% agarose gel electrophoresis. The band of interest wascleaved from the gel, followed by isolation and purification with theuse of the DNA gel extraction kit (Qiagen) in accordance with theattached protocols. The resultant was inserted between the NdeI site andthe NotI site of pET-22b(+) (Novagen), so as to construct the 4E6tetramer comprising Strep-tag at its N terminus, four VHH monomerslinked with each other with three linker peptides (GGSGG)₂ (SEQ ID NO:28) (FIG. 12c ).

(2) Expression of the Recombinant Proteins in E. coli

The 4E6 tetramer, the 4E6 dimer toxin, and the 4E6 dimer EGFP genesobtained as described above were inserted between the NdeI site and theNotI site of pET22b(+). This expression vector (plasmid DNA) wasintroduced into E. coli RosettaGami (DE3)2 (Novagen), recombinant E.coli cells were cultured using 200 ml of 2YT medium containing 100 μg/mlampicillin (Sigma-Aldrich) at 37° C. in accordance with the attachedinstructions, and 1 mM IPTG (isopropyl-β-thiogalactopyranoside, TAKARABIO INC.) was added when OD₆₀₀ reached 0.4 to 0.5, followed bycultivation at 30° C. for 3 hours. Following the cultivation, E. colicells were recovered and resuspended in 20 ml of extraction buffer (50mM Na phosphate, pH 7.8, 300 mM NaCl, EDTA-free protease inhibitorcocktail, Roche), and the cells were disrupted by ultrasonication on iceusing Sonifier 250 (Branson) at the output control of 2 and the dutycycle of 80% for 1 minute twice. After the treatment, the suspension wascentrifuged at 15,000 rpm and 4° C. for 20 minutes, and the supernatantwas recovered. The fusion protein was purified using the HisTrap column(GE Healthcare). The centrifuged supernatant of the ultrasonicatedsuspension was directly applied to the HisTrap column, the column waswashed with a binding buffer (20 mM Na phosphate, 0.5 M NaCl, 20 mMimidazole, pH 7.4), and the fusion protein was then eluted with aneluate containing 500 mM imidazole. The purified sample was subjected to10% SDS-polyacrylamide gel electrophoresis and stained with CBB. Thepurified recombinant protein was observed, and the concentration of thetarget protein band was estimated by comparing with the results ofstaining of BSA (bovine serum albumin) (FIG. 13a-c ). The estimatedconcentrations of the 4E6 dimer toxin, the 4E6 dimer EGFP, and the 4E6tetramer were 0.8 mg/ml, 1.2 mg/ml, and 1.6 mg/ml, respectively.

Example 7: Measurement of Activity of 4E6 Dimer Toxin and 4E6 Tetramerto Induce Cancer Cell Apoptosis

Whether or not the 4E6 dimer toxin fusion protein exerts activity ofinducing apoptosis on cultured hTRAIL-R2-expressing human cancer cells(Colo205, obtained from American Type Culture Collection) wasinvestigated. The cells were added to a Falcon 96-well culture plate(Becton, Dickinson and Company) at 5×10⁴ cells/50 μl of medium (RPMI1640 medium containing 0.2% fetal bovine serum (Tissue CultureBiologicals))/well, the cells were cultured for 2 hours, and then 50 μleach of media containing the 4E6 dimer toxin and the 4E6 dimer EGFPpurified as described above was added to the final concentrations of2,000, 800, 160, 32, 6.4, 1.28, 0.256, and 0.0512 ng/ml (2×finalconcentration), respectively. 10 μl of an MTT reagent (Nacalai Tesque,Inc.) was added, after cultivating for 2 days. The cells were thencultured for an additional 2 hours, a solubilizer was added at 100μl/well, the cells were lysed by pipetting, and the absorbance at OD₅₇₀nm was measured. The measured value of a cell-free medium in a well wassubtracted as a background value from all the measured values. Thevalues measured for the wells containing cells alone were designated100%, and the relative values were determined. Assay was performed intriplicate, and an average value of the results for 3 wells wasemployed. As shown in FIG. 14a , a fusion construct comprising ananti-TRAIL-R2 VHH antibody dimer and Toxin; that is, Immunotoxin, didnot induce apoptosis at all.

Whether or not the 4E6 dimer binds to the cells was examined asdescribed below. Specifically, human colon cancer cells (Colo205 cells)and pancreatic cancer cells (BxPC-3 cells) (2×10⁵ each) were reacted in50 μl of phosphate buffered saline (pH 7.4) containing 10 μg/ml 4E6dimer EGFP and 2% fetal bovine serum on ice for 30 minutes. The cellswere washed twice with the serum-containing phosphate buffered saline,and the fluorescent intensity of the cells was analyzed using theFACSVerse flow cytometer (BD Biosciences). Both cells were stained withthe 4E6 dimer EGFP. In contrast, these cells were not stained with thecontrol dimer EGFP (FIG. 15). Accordingly, the 4E6 dimer is consideredto be able to bind to the cells.

According to the above results, the 4E6 dimer toxin binds to TRAIL-R2 ona cell membrane, but it does not allow TRAIL-R2 molecules to aggregatein a trimer or larger multimer, and the 4E6 dimer toxin is notincorporated into cells. Accordingly, the 4E6 dimer toxin was notconsidered to be able to induce apoptosis of Colo205 cells.

In order to examine whether or not the 4E6 monomer and the 4E6 tetramerexerts activity of inducing apoptosis, subsequently, 50 μl each of mediacontaining the 4E6 monomer and the 4E6 tetramer purified as describedabove was added at final concentrations of 25,000, 5,000, 1,000, 200,40, 8, 1.6, and 0.32 pg/ml (2×final concentration). As a result, the 4E6monomer did not induce apoptosis, but the 4E6 tetramer having activityof aggregating TRAIL-R2 molecules in a trimer or larger multimer inducedstrong apoptosis of Colo205 cells (FIG. 14b ).

The above results demonstrate that the anti-hTRAIL-R VHH antibodytetramer having activity of aggregating TRAIL-R molecules in a trimer orlarger multimer induces cancer cell apoptosis more efficiently than theanti-hTRAIL-R VHH antibody dimer toxin.

Example 8: Activity of 4E6 Tetramer Expressed in E. coli to InduceApoptosis of Human Colon Cancer Cells and Pancreatic Cancer Cells

Activity of the 4E6 tetramer to induce cancer cell apoptosis of humancolon cancer cells (Colo205 cells) and pancreatic cancer cells (BxPC-3cells) (obtained from American Type Culture Collection) was examined.The cells were suspended in RPMI 1640 medium (Sigma) supplemented with10% fetal bovine serum (Tissue Culture Biologicals), the cells wereadded to a Falcon 96-well culture plate (Becton, Dickinson and Company)at 3×10³ cells/50 μl/well, the cells were cultured overnight, and 50 μleach of media containing the 4E6 tetramer or hTRAIL (Wako Pure ChemicalIndustries, Ltd.) was then added. Colo205 cells were cultured overnightfor a day, BxPC-3 cells were overnight for two days, 10 μl of a viablecell count reagent SF (Nacalai Tesque, Inc.) was added, the cells werecultured for an additional 4 to 6 hours, and the absorbance at 450 nmwas then measured. The measured value of a cell-free medium in a wellwas subtracted as a background value. The values measured for the wellscontaining cells alone were designated 100%, and the relative valueswere determined. The results were shown as the mean plus the standarddeviation of the results for 3 wells at each concentration. As shown inFIG. 16, the 4E6 tetramer induced apoptosis of Colo205 and BxPC-3 cellsin a concentration-dependent manner, and the IC₅₀ values were 2 pmol/land 8 pmol/l, respectively. The IC₅₀ value of hTRAIL prepared using E.coli was 400 pmol/l, and the 4E6 tetramer was found to be able to induceapoptosis at a concentration lower than that of hTRAIL.

Example 9: Expression and Secretion of 4E6 Tetramer and 4E6 Dimer EGFPin Bifidobacterium and Purification Thereof (1) Preparation ofRecombinant Bifidobacterium Via Electroporation

The vector gene cassette for secretion and expression of the 4E6tetramer and the 4E6 dimer EGFP in Bifidobacterium (see Example 1) wasinserted between HindIII and NotI of the pKKT427 vector (Yasui K., etal., Nucleic Acids Res., 2009), and the resultant was introduced into B.longum 105-A via electroporation. Electroporation was carried out underconditions of 2.4 kV, 25 μF, and 200 ohms.

(2) Purification of 4E6 Tetramer and 4E6 Dimer EGFP Expressed andSecreted in Bifidobacterium

The recombinant Bifidobacterium obtained in the above (1) was added toMRS liquid medium (Lactobacilli MRS Broth, Difco Laboratories, Detroit,Mich.) containing 100 μg/ml spectinomycin supplemented with 50 mMsucrose, 3.4 mg/ml L-ascorbic acid sodium salt, and 0.2 mg/ml L-cysteinehydrochloride, and the cells were cultured anaerobically overnight. Thecells were cultured anaerobically with the use of a sealed containercontaining a deoxidizer, Anaero Pack Kenki (Mitsubishi Gas ChemicalCompany, Inc., Tokyo, Japan). After cultivating overnight, theabsorbance at 600 nm the culture solution was measured, and the culturesolution was added to a fresh liquid medium, so as to adjust theabsorbance to 0.1. The cells were cultured anaerobically for 6 to 7hours, the culture supernatant was collected by centrifugation at 4° C.and 9,400×g for 10 minutes. The recombinant protein was purified usingthe HisTrap column (GE Healthcare). The culture supernatant was appliedto the HisTrap column, the column was washed with a binding buffer (50mM Na phosphate, 0.3 M NaCl, 20 mM imidazole, pH 7.8), and the proteinwas then eluted with an eluate containing 500 mM imidazole. The purifiedprotein obtained from 1 ml of the culture supernatant was subjected toSDS polyacrylamide gel electrophoresis and then stained with OrioleFluorescent Gel Stain (Bio-Rad) (FIG. 17). Both the 4E6 tetramer and the4E6 dimer EGFP were detected around the deduced molecular weight (about60 kDa). The amount of the 4E6 tetramer secreted in the culturesupernatant was estimated to be 400 ng/ml, and that of the 4E6 dimerEGFP was estimated to be 3.2 ng/ml. The results demonstrate that boththe recombinant proteins were secreted and that the 4E6 tetramer wasmore efficiently expressed and secreted than the 4E6 dimer EGFP.

Example 10: Activity of Anti-hTRAIL-R2 VHH Antibody Tetramer (4E6Tetramer) Expressed and Secreted in Bifidobacterium to Induce CancerCell Apoptosis

Activity of the 4E6 tetramer to induce cancer cell apoptosis of humancolon cancer cells (Colo205 cells) (American Type Culture Collection)was examined. The cells were suspended in RPMI 1640 medium (Sigma)supplemented with 10% fetal bovine serum (Tissue Culture Biologicals),and the cells were added to a Falcon 96-well culture plate (Becton,Dickinson and Company) at 3×10³ cells/50 μl medium/well. The cells werecultured overnight, and 50 μl each of media containing the 4E6 tetrameror TRAIL at the final concentration of 0.3 pM to 10 nM was addedthereto. The cells were further cultured overnight, 10 μl of a viablecell count reagent SF (Nacalai Tesque, Inc.) was added thereto, thecells were cultured for 6 hours, and the absorbance at 450 nm wasmeasured. The measured value of a cell-free medium in a well wassubtracted as a background value. The values measured for the wellscontaining cells alone were designated 100%, and the relative valueswere determined. The results were shown as the mean plus the standarddeviation of the results for 3 wells at each concentration. As shown inFIG. 18, the 4E6 tetramer inhibited the growth of the Colo205 cells in aconcentration-dependent manner, and the IC₅₀ value was 0.02 nmol/l.Meanwhile, the IC₅₀ value of hTRAIL (Wako Pure Chemical Industries,Ltd.) prepared in E. coli was 0.4 nmol/l. Thus, the 4E6 tetramerexpressed and secreted in Bifidobacterium was found to have higheractivity to induce apoptosis than hTRAIL.

Example 11: Examination of Antitumor Effect of Recombinant B. longum ViaIntravenous Administration in Xenograft Model Transplanted with Colo205Cells

Antitumor effects of recombinant B. longum 105-A cells expressing andsecreting the anti-hTRAIL-R2 VHH antibody tetramer (4E6 tetramer) wereexamined when the recombinant B. longum 105-A cells were administeredintravenously to xenograft models prepared by transplanting human coloncancer cells (Colo205 cells) subcutaneously into nude mice to form solidcancer. Specifically, 2×10⁶ Colo205 cells were transplantedsubcutaneously into 6-week-old female KSN/Slc nude mice, and 9 dayslater, the mice were divided into groups (n=6; control, 4E6 tetramer,and 4E6 dimer EGFP groups) so as to adjust the tumor mass volume of eachgroup to approximately 280 mm³, and the recombinant Bifidobacteriumprepared in accordance with Example 9 were administered intravenously at1.5×10⁹ cells/mouse. The B. longum 105-A used was prepared viacentrifugation and resuspension with saline. For nutritionalsupplementation of B. longum 105-A in the body, 1 ml of 20% lactulosewas intraperitoneally administered every day. The tumor size wasmeasured with the use of a caliper 0, 2, 4, 7, 11, 14, 18, and 21 daysafter Bifidobacterium administration. The tumor volume was determined bythe formula: (shorter diameter)²×(longer diameter)/2. The results areshown in FIG. 19. In comparison with the control group, the tumor growthwas inhibited by 51% 21 days after administrating the recombinantBifidobacterium expressing and secreting the 4E6 tetramer. On the otherhand, the inhibitory effects of tumor growth were not observed, in thenegative control group to which Bifidobacterium secreting the 4E6 dimerEGFP had been administered.

Together with measuring the tumor size, body weights of all groups weremeasured at 0, 2, 4, 7, 11, 14, 18, and 21 days after Bifidobacteriumadministration. As shown in FIG. 20, body weight loss was not detectedin the 4E6 tetramer group, in comparison with the control group and the4E6 dimer EGFP group. On the basis of the results described above, the4E6 tetramer is considered to inhibit tumor growth by apoptosis-inducingactivity without causing side effects such as body weight loss.

Example 12: Examination of Antitumor Effect of Recombinant B. longum ViaIntravenous Administration in Xenograft Model Transplanted with BxPC-3Cells

Antitumor effects of recombinant B. longum 105-A cells expressing andsecreting the anti-hTRAIL-R2 VHH antibody tetramer (4E6 tetramer) wereexamined when the recombinant B. longum 105-A cells were administeredintravenously to xenograft models prepared by transplanting humanpancreatic cancer cells (BxPC-3 cells) subcutaneously into nude mice toform solid cancer. Specifically, 2×10⁶ BxPC-3 cells were transplantedsubcutaneously into 8-week-old female KSN/Slc nude mice, and 8 dayslater, the mice were divided into groups (n=6; control, 4E6 tetramer,and 4E6 dimer EGFP groups) so as to adjust the tumor mass volume of eachgroup to approximately 230 mm³, and the recombinant Bifidobacteriumcells prepared in accordance with Example 9 were administeredintravenously at 1.5×10⁹ cells/mouse. The B. longum 105-A used wasprepared via centrifugation and resuspension with saline. Fornutritional supplementation of B. longum 105-A in the body, 1 ml of 20%lactulose was intraperitoneally administered every day. The tumor sizewas measured with the use of a caliper 0, 3, 6, 10, 13, and 17 daysafter Bifidobacterium administration. The tumor volume was determined bythe formula: (shorter diameter)²×(longer diameter)/2. The results areshown in FIG. 21. The tumor growth was inhibited by 52% 17 days afteradministrating the recombinant Bifidobacterium expressing and secretingthe 4E6 tetramer, in comparison with the control group. The inhibitoryeffects of tumor growth were not observed in the negative control groupto which Bifidobacterium secreting the 4E6 dimer EGFP had beenadministered.

Together with measuring the tumor size, body weights of all groups weremeasured at 0, 3, 6, 10, 13, and 17 days after Bifidobacteriumadministration. As shown in FIG. 22, body weight loss was not detectedin the 4E6 tetramer group, in comparison with the control group and the4E6 dimer EGFP group. On the basis of the results described above, the4E6 tetramer is considered to inhibit tumor growth by apoptosis-inducingactivity without causing side effects such as body weight loss.

Example 13: Preparation of 4P6 Trimer Recombinant E. coli BL21 (DE3) andExpression and Purification of Recombinant Proteins

(1) Preparation of Gene Expression Cassette of Anti-hTRAIL-R1 VHHAntibody Trimer (4P6 Trimer) to be Expressed in E. coli

A gene encoding a 4P6 trimer comprising Strep-tag at its N terminus andthe His-Tag sequence at its C terminus and 3 monomers of theanti-hTRAIL-R1 VHH antibody obtained in accordance with Example 4 (1)linked with two linker peptides (GGSGG)₂ (SEQ ID NO: 28) was insertedbetween the NdeI site and the NotI site of pET-22b(+) (Novagen), so asto construct a 4P6 trimer gene expression cassette to be expressed in E.coli (see Example 6).

(2) Expression of Recombinant Protein in E. coli

The plasmid vector prepared as described above was introduced into E.coli BL21Star™ (DE3) One Shot (Life Technologies). In accordance withthe attached instructions, recombinant E. coli cells were cultured in200 ml of 2YT medium containing 100 μg/ml ampicillin (Sigma-Aldrich) at37° C., and 1 mM IPTG (isopropyl-β-thiogalactopyranoside, TAKARA BIOINC.) was added thereto when OD₆₀₀ reached 0.4 to 0.5, followed bycultivation at 30° C. for 3 hours. Following the cultivation, E. colicells were recovered and resuspended in 20 ml of an extraction buffer(50 mM Na phosphate, pH 7.8, 300 mM NaCl, EDTA-free protease inhibitorcocktail, Roche). The cells were disrupted by ultrasonication on iceusing Sonifier 250 (Branson) at the output control of 2 and the dutycycle of 80% for 1 minute twice. The supernatant was recovered bycentrifuging the treated suspension at 9,400×g and 4° C. for 20 minutes.The fusion protein was purified with the use of the HisTrap column (GEHealthcare, U.K.). The centrifuged supernatant of the ultrasonicatedsuspension was applied directly to the HisTrap column, the column waswashed with a binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mMimidazole, pH 7.4), and the fusion protein was then eluted with the useof an eluate containing 500 mM imidazole. Further, the elusion fractionwas applied to the Strep-Tactin column (IBA) and purified in accordancewith the attached instructions. The purified protein in an amountequivalent to 50 ng of BSA was subjected to SDS polyacrylamide gelelectrophoresis and then stained with Oriole Fluorescent Gel Stain(Bio-Rad) (FIG. 23). The 4P6 trimer was detected around the deducedmolecular weight (about 42 kDa). The results demonstrate that the 4P6trimer can be expressed in E. coli and purified.

Example 14: Measurement of Activity of 4P6 Trimer Expressed in E. colito Induce Cancer Cell Apoptosis

Activity of the 4P6 trimer to induce cancer cell apoptosis of humancolon cancer cells (Colo205 cells) (American Type Culture Collection)was examined. The cells were suspended in RPMI 1640 medium (Sigma)supplemented with 10% fetal bovine serum (Tissue Culture Biologicals),and the cells were added to a Falcon 96-well culture plate (Becton,Dickinson and Company) at 3×10³ cells/50 μl medium/well. The cells werecultured overnight, and 50 μl each of media containing the 4P6 trimer atthe final concentration of 10 pM to 10 nM was added thereto. The cellswere further cultured overnight for two days, 10 μl of a viable cellcount reagent SF (Nacalai Tesque, Inc.) was added thereto, the cellswere cultured for 4 hours, and the absorbance at 450 nm was measured.The measured value of a cell-free medium in a well was subtracted as abackground value. The values measured for the wells containing cellsalone were designated 100%, and the relative values were determined. Thevalues were shown as the mean plus the standard deviation of the resultsfor 3 wells at each concentration. As shown in FIG. 24, the 4P6 trimerinhibited the growth of the Colo205 cells in a concentration-dependentmanner, and the IC₅₀ value was 0.4 nmol/1. The IC₅₀ value of hTRAILprepared in E. coli (Wako Pure Chemical Industries, Ltd.) was 0.4nmol/1, and the 4P6 trimer expressed in E. coli exerted activity ofinducing apoptosis at the similar level as that of hTRAIL.

Example 15: Expression and Secretion of Anti-hTRAIL-R1 VHH AntibodyTrimer (4P6 Trimer) in Bifidobacterium and Purification Thereof (1)Preparation of Recombinant Bifidobacterium Via Electroporation

The vector gene cassette for expression and secretion of the 4P6 trimerin Bifidobacterium, which was prepared by replacing the 4E6 tetramerportion in the expression cassette prepared in Example 1 (1) with the4P6 trimer obtained in accordance with Example 4 (1), was insertedbetween HindIII and NotI of the pKKT427 vector (Yasui K., et al.,Nucleic Acids Res., 2009), and the resultant was introduced into B.longum 105-A via electroporation. Electroporation was carried out underconditions of 2.4 kV, 25 μF, and 200 ohms.

(2) Purification of 4P6 Trimer Expressed and Secreted in Bifidobacterium

The recombinant Bifidobacterium obtained in the above (1) was added toMRS liquid medium containing 100 μg/ml spectinomycin (Lactobacilli MRSBroth, Difco Laboratories, Detroit, Mich.) supplemented with 50 mMsucrose, 3.4 mg/ml L-ascorbic acid sodium salt, and 0.2 mg/ml L-cysteinehydrochloride, and the cells were cultured anaerobically overnight. Thecells were cultured anaerobically with the use of a sealed containercontaining a deoxidizer, Anaero Pack Kenki (Mitsubishi Gas ChemicalCompany, Inc., Tokyo, Japan). Following cultivating overnight, theabsorbance at 600 nm of the culture solution was measured, and theculture solution was added to a fresh liquid medium, so as to adjust theabsorbance to 0.1. The cells were cultured anaerobically for 7 hours,and the culture supernatant was then collected by centrifuging theculture medium at 4° C. and 9,400×g for 10 minutes. The recombinantprotein was purified using the HisTrap column (GE Healthcare). Theculture supernatant was applied to the HisTrap column, the column waswashed with a binding buffer (50 mM Na phosphate, 0.3 M NaCl, 20 mMimidazole, pH 7.8), and the protein was then eluted with an eluatecontaining 500 mM imidazole. The purified protein obtained from 0.6 mlof the culture supernatant was subjected to SDS polyacrylamide gelelectrophoresis and then stained with Oriole Fluorescent Gel Stain(Bio-Rad) (FIG. 25). The 4P6 trimer was detected around the deducedmolecular weight (approximately 42 kD). As a result of quantificationwith the use of BSA, the amount of the 4P6 trimer secreted in theculture supernatant was estimated to be 30 ng/ml. The resultsdemonstrate that the 4P6 trimer would be expressed and secreted inBifidobacterium.

Example 16: Activity of Anti-hTRAIL-R1 VHH Antibody Trimer (4P6 Trimer)Expressed and Secreted in Bifidobacterium to Induce Cancer CellApoptosis

Activity of the 4P6 trimer to induce cancer cell apoptosis of humancolon cancer cells (Colo205 cells) (American Type Culture Collection)and pancreatic cancer cells (BxPC-3 cells) (American Type CultureCollection) was examined. The cells were suspended in RPMI 1640 medium(Sigma) supplemented with 10% fetal bovine serum (Tissue CultureBiologicals), and the cells were added to a Falcon 96-well culture plate(Becton, Dickinson and Company) at 3×10³ cells/50 μl medium/well. Thecells were cultured overnight, and 50 μl each of media containing the4P6 trimer at the final concentration of 0.3 pM to 10 nM was addedthereto. The cells were further cultured overnight for two days, 10 μlof a viable cell count reagent SF (Nacalai Tesque, Inc.) was addedthereto, the cells were cultured for 2 hours, and the absorbance at 450nm was measured. The measured value of a cell-free medium in a well wassubtracted as a background. The values measured for the wells containingcells alone were designated 100%, and the relative values weredetermined. The results were shown as the mean plus the standarddeviation of the results for 3 wells at each concentration. As shown inFIG. 26a , the 4P6 trimer inhibited the growth of Colo205 cells in aconcentration-dependent manner, and the IC₅₀ value was 0.08 nmol/l. TheIC₅₀ value of hTRAIL (Wako Pure Chemical Industries, Ltd.) prepared inE. coli was 0.4 nmol/1, and the 4P6 trimer expressed and secreted inBifidobacterium was found to have higher activity to induce apoptosisthan hTRAIL. As shown in FIG. 26b , the 4P6 trimer also inhibited thegrowth of BxPC-3 cells in a concentration-dependent manner

Example 17: Examination of Antitumor Effect of Recombinant B. longumExpressing and Secreting 4P6 Trimer Via Intravenous Administration inXenograft Model Transplanted with BxPC-3-Luc#2 Cells

Antitumor effects of recombinant B. longum 105-A cells expressing andsecreting the anti-hTRAIL-R1 VHH antibody trimer (4P6 trimer) wereexamined when the recombinant B. longum 105-A cells were administeredintravenously to xenograft models prepared by transplanting humanpancreatic cancer cells (BxPC-3-Luc#2 cells, obtained from JCRB CellBank) subcutaneously into nude mice to form solid cancer. Specifically,3×10⁶ BxPC-3-Luc#2 cells were transplanted subcutaneously into6-week-old female KSN/Slc nude mice, and 15 days later, the mice weredivided into groups (n=5; the control, the 4P6 trimer, and the pKKT427vector groups) so as to adjust the tumor mass volume of each group toapproximately 135 mm³, and the recombinant Bifidobacterium cellsprepared in accordance with Example 15 were administered intravenouslyat 3×10⁸ cells/mouse. The B. longum 105-A used was prepared viacentrifugation and resuspension with saline. For nutritionalsupplementation of B. longum 105-A in the body, 1 ml of 20% lactulosewas intraperitoneally administered every day. The tumor size wasmeasured with the use of a caliper 15, 19, 21, 24, 28, 31, and 35 daysafter tumor transplantation. The tumor volume was determined by theformula: (shorter diameter)²×(longer diameter)/2. The results are shownin FIG. 27. The tumor growth was inhibited by 65% 20 days afteradministrating the recombinant Bifidobacterium expressing and secretingthe 4P6 trimer, in comparison with the control group. The inhibitoryeffects of tumor growth were not observed in the negative control groupto which pKKT427-introduced Bifidobacterium had been administered.

Together with measuring the tumor size, body weights of all groups weremeasured at 15, 19, 21, 24, 28, 31, and 35 days after tumortransplantation. As shown in FIG. 28, body weight loss was not detectedin the 4P6 trimer group in comparison with the control group and thepKKT427-introduced Bifidobacterium group. On the basis of the resultsdescribed above, the 4P6 trimer is considered to inhibit tumor growth byapoptosis-inducing activity without causing side effects such as bodyweight loss.

INDUSTRIAL APPLICABILITY

According to the present invention, cancer cell apoptosis can beeffectively induced via topical application of the anti-hTRAIL-R1antibody(ies) and the anti-hTRAIL-R2 antibody(ies) having potentagonistic activity into the tumor site while reducing the toxicityimposed on normal cells.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. A method of treating a tumor, comprising administering recombinant bacteria of the genus Bifidobacterium to a subject in need thereof by an intravenous route; wherein the recombinant bacteria comprise a nucleic acid encoding a fusion protein, the fusion protein comprising a signal peptide and 3 or more anti-TRAIL-R1 single-chain antibodies and/or 3 or more anti-TRAIL-R2 single-chain antibodies, in an expressible state.
 2. The method of claim 1, wherein each of the 3 or more anti-TRAIL-R1 single-chain antibodies comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the CDR2 comprises the amino acid sequence of SEQ ID NO: 23; and the CDR3 comprises the amino acid sequence of SEQ ID NO:
 24. 3. The method of claim 1, wherein each of the 3 or more anti-TRAIL-R2 single-chain antibodies comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 15, the CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the CDR3 comprises the amino acid sequence of SEQ ID NO:
 17. 4. The method of claim 1, wherein the fusion protein further comprises one or more functional peptides.
 5. The method of claim 4, wherein the functional peptides comprise a labeling protein.
 6. The method of claim 1, wherein the tumor is a colon cancer or a pancreatic cancer.
 7. The method of claim 1, where the recombinant Bifidobacterium expresses and secretes a 4E6 tetramer.
 8. The method of claim 1, wherein the subject is a mammal. 